Methods for generating high titer helper-free preparations of released recombinant AAV vectors

ABSTRACT

This invention provides methods and compositions for producing high titer, substantially purified preparations of recombinant adeno-associated virus (AAV) that can be used as vectors for gene delivery. At the onset of vector production, AAV producer cells of this invention typically comprise one or more AAV packaging genes, an AAV vector comprising a heterologous (i.e. non-AAV) transgene of interest, and a helper virus such as an adenovirus. The AAV vector preparations produced are generally replication incompetent but are capable of mediating delivery of a transgene of interest (such as a therapeutic gene) to any of a wide variety of tissues and cells. The AAV vector preparations produced according to this invention are also substantially free of helper virus as well as helper viral and cellular proteins and other contaminants. The invention described herein provides methods of producing rAAV particles by culturing producer cells under conditions, such as temperature and pH, that promote release of virus. Also provided is a quantitative, high-throughput assay useful in the assessment of viral infectivity and replication, as well as in the screening of agent that affect viral infectivity and/or replication.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. Ser. No. 09/142,474, filed Sep.4, 1998, which was a U.S. National filing under Section 371 (via PCTapplication PCT/US98/18600, filed Sep. 4, 1998), which claims thepriority benefit of provisional U.S. Ser. Nos. 60/071,733, filed Jan.16, 1998, and 60/084,193, filed Sep. 5, 1997. This application alsoclaims the priority benefit of PCT patent application PCT/US99/20524filed Sep. 7, 1999, designating the U.S., which claims priority toprovisional application U.S. Ser. No. 60/123,685, filed Mar. 10, 1999and U.S. Ser. No. 09/142,474, filed Sep. 4, 1998. All of theseapplications are incorporated by reference in their entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made in part during work supported by a grant fromthe National Institutes of Health (NIH) R44DK4460. The government mayhave certain rights in this invention.

TECHNICAL FIELD

The present invention relates generally to the field of recombinantadeno-associated virus (AAV) vectors and preparations thereof that canbe used for gene transfer. More specifically, it relates to methods forgenerating high titer preparations of recombinant AAV vectors that aresubstantially free of helper virus (e.g. adenovirus) as well as cellularproteins.

BACKGROUND ART

Adeno-associated viruses (AAV) have unique features that make themattractive as vectors for gene therapy. Adeno-associated viruses infecta wide range of cell types. However, they are non-transforming, and arenot implicated in the etiology of any human disease. Introduction of DNAto recipient host cells generally leads to long-term persistence andexpression of the DNA without disturbing the normal metabolism of thecell.

There are at least three desirable features of a recombinant AAV vectorpreparation for use in gene transfer, especially in human gene therapy.First, it is preferred that the vector should be generated at titerssufficiently high to transduce an effective proportion of cells in thetarget tissue. Gene therapy in vivo typically requires a high number ofvector particles. For example, some treatments may require in excess of10⁸ particles, and treatment of cystic fibrosis by direct delivery tothe airway may require in excess of 10¹⁰ particles. Second, it ispreferred that the vector preparations should be essentially free ofreplication-competent AAV (i.e. phenotypically wild-type AAV which canbe replicated in the presence of helper virus or helper virusfunctions). Third, it is preferred that the rAAV vector preparation as awhole be essentially free of other viruses (such as a helper virus usedin AAV production) as well as helper virus and cellular proteins, andother components such as lipids and carbohydrates, so as to minimize oreliminate any risk of generating an immune response in the context ofgene therapy. This latter point is especially significant in the contextof AAV because AAV is a “helper-dependent” virus that requiresco-infection with a helper virus (typically adenovirus) or otherprovision of helper virus functions in order to be effectivelyreplicated and packaged during the process of AAV production; and,moreover, adenovirus has been observed to generate a host immuneresponse in the context of gene therapy applications (see, e.g., Byrneset al., Neuroscience 66:1015, 1995; McCoy et al., Human Gene Therapy6:1553, 1995; and Barr et al., Gene Therapy 2:151, 1995). The methods ofthe present invention address these and other desirable features of rAAVvector preparations, as described and illustrated in detail below.

General reviews of AAV virology and genetics are available elsewhere.The reader may refer inter alia to Carter, “Handbook of Parvoviruses”,Vol. I, pp. 169-228 (1989), and Berns, “Virology”, pp. 1743-1764, RavenPress, (1990). AAV is a replication-defective virus, which means that itrelies on a helper virus in order to complete its replication andpackaging cycle in a host cell. Helper viruses capable of supporting AAVreplication are exemplified by adenovirus, but include other virusessuch as herpes and pox viruses. The AAV genome generally comprises thepackaging genes rep and cap, with other necessary functions beingprovided in trans from the helper virus and the host cell.

AAV particles are comprised of a proteinaceous capsid having threecapsid proteins, VP1, VP2 and VP3, which enclose a ˜4.6 kb linearsingle-stranded DNA genome. Individual particles package only one DNAmolecule strand, but this may be either the plus or minus strand.Particles containing either strand are infectious, and replicationoccurs by conversion of the parental infecting single strand to a duplexform, and subsequent amplification, from which progeny single strandsare displaced and packaged into capsids. Duplex or single-strand copiesof AAV genomes (sometimes referred to as “proviral DNA” or “provirus”)can be inserted into bacterial plasmids or phagemids, and transfectedinto adenovirus-infected cells.

By way of illustration, the linear genome of serotype AAV2 is terminatedat either end by an inverted terminal repeat (ITR) sequence. Between theITRs are three transcription promoters p5, p19, and p40 that are used toexpress the rep and cap genes (Laughlin et al., 1979, Proc. Natl. Acad.Sci. USA, 76:5567-5571). ITR sequences are required in cis and aresufficient to provide a functional origin of replication, integrationinto the cell genome, and efficient excision and rescue from host cellchromosomes or recombinant plasmids. The rep and cap gene productsprovide functions for replication and encapsidation of viral genome,respectively, and it is sufficient for them to be present in trans.

The rep gene is expressed from two promoters, p5 and p19, and producesfour proteins designated Rep78, Rep68, Rep52 and Rep40. Only Rep78 andRep68 are required for AAV duplex DNA replication, but Rep52 and Rep40appear to be needed for progeny, single-strand DNA accumulation(Chejanovsky et al., Virology 173:120, 1989). Rep68 and Rep78 bindspecifically to the hairpin conformation of the AAV ITR and possessseveral enzyme activities required for resolving replication at the AAVtermini. Rep78 and Rep68, also exhibit pleiotropic regulatory activitiesincluding positive and negative regulation of AAV genes and expressionfrom some heterologous promoters, as well as inhibitory effects on cellgrowth. The cap gene encodes capsid proteins VP1, VP2, and VP3. Theseproteins share a common overlapping sequence, but VP1 and VP2 containadditional amino terminal sequences transcribed from the p40 promoter byuse of alternate initiation codons. All three proteins are required foreffective capsid production.

AAV genomes have been introduced into bacterial plasmids by proceduressuch as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA,79:2077-2081), addition of synthetic linkers containing restrictionendonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) orby direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem.,259:4661-4666). Transfection of such AAV recombinant plasmids intomammalian cells with an appropriate helper virus results in rescue andexcision of the AAV genome free of any plasmid sequence, replication ofthe rescued genome and generation of progeny infectious AAV particles.

Recombinant AAV vectors comprising a heterologous polynucleotide oftherapeutic interest may be constructed by substituting portions of theAAV coding sequence in bacterial plasmids with the heterologouspolynucleotide. General principles of rAAV vector construction are alsoreviewed elsewhere. See, e.g., Carter, 1992, Current Opinions inBiotechnology, 3:533-539; and Muzyczka, 1992, Curr. Topics in Microbiol.and Immunol., 158:97-129). The AAV ITRs are generally retained, sincepackaging of the vector requires that they be present in cis. However,other elements of the AAV genome, in particular, one or more of thepackaging genes, may be omitted. The vector plasmid can be packaged intoan AAV particle by supplying the omitted packaging genes in trans via analternative source.

In one approach, the sequence flanked by AAV ITRs (the rAAV vectorsequence), and the AAV packaging genes to be provided in trans, areintroduced into the host cell in separate bacterial plasmids. Examplesof this approach are described in Ratschin et al., Mol. Cell. Biol.4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466(1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin etal., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell.Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828)have described a packaging plasmid called pAAV/Ad, which consists of Repand Cap encoding regions enclosed by ITRs from adenovirus. Human airwayepithelial cells from a cystic fibrosis patient have been transducedwith an AAV vector prepared using the pAAV/Ad packaging plasmid and aplasmid comprising the selective marker gene neo expressed via the AAVp5 promoter (Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349,1992).

A second approach is to provide either the vector sequence, or the AAVpackaging genes, in the form of an episomal plasmid in a mammalian cellused for AAV replication. For example, U.S. Pat. No. 5,173,414 describesa cell line in which the vector sequence is present as a high-copyepisomal plasmid. The cell lines can be transduced with thetrans-complementing AAV functions rep and cap to generate preparationsof AAV vector. This approach is not ideal, because the copy number percell cannot be rigorously controlled and episomal DNA is much morelikely to undergo rearrangement, leading to production of vectorbyproducts.

A third approach is to provide either the vector sequence, or the AAVpackaging genes, or both, stably integrated into the genome of themammalian cell used for replication.

One exemplary technique is outlined in international patent applicationWO 95/13365 (Targeted Genetics Corporation and Johns Hopkins University)and corresponding U.S. Pat. No. 5,658,776 (by Flotte et al.). Thisexample uses a mammalian cell with at least one intact copy of a stablyintegrated rAAV vector, wherein the vector comprises an AAV ITR and atranscription promoter operably linked to a target polynucleotide, butwherein the expression of rep is limiting. In a preferred embodiment, anAAV packaging plasmid comprising the rep gene operably linked to aheterologous AAV is introduced into the cell, and then the cell isincubated under conditions that allow replication and packaging of theAAV vector sequence into particles.

A second exemplary technique is outlined in patent application WO95/13392 (Trempe et al.). This example uses a stable mammalian cell linewith an AAV rep gene operably linked to a heterologous promoter so as tobe capable of expressing functional Rep protein. In various preferredembodiments, the AAV cap gene can be provided stably as well or can beintroduced transiently (e.g. on a plasmid). A recombinant AAV vector canalso be introduced stably or transiently.

Another exemplary technique is outlined in patent application WO96/17947 (by Targeted Genetics Corporation, J. Allen). This example usesa mammalian cell which comprises a stably integrated AAV cap gene, and astably integrated AAV rep gene operably linked to a heterologouspromoter and inducible by helper virus. In various preferredembodiments, a plasmid comprising the vector sequence is also introducedinto the cells (either stably or transiently). The rescue of AAV vectorparticles is then initiated by introduction of the helper virus.

Other methods for generating high-titer preparations of recombinant AAVvectors have been described. International Patent Application No.PCT/US98/18600 describes culturing a cell line which can produce rAAVvector upon infection with a helper virus; infecting the cells with ahelper virus, such as adenovirus; and lysing the cells. AAV and otherviral production methods and systems are also described in, for example,WO 97/09441 (PCT/US96/14423);. WO 97/08298 (PCT/US96/13872); WO 97/21825(PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; Perrin etal. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132.

These various examples address the issue of providing AAV atsufficiently high titer, minimizing recombination between vector andpackaging components, and reducing or avoiding the potentialdifficulties associated with the expression of the AAV rep gene inmammalian cell line (since the Rep proteins can not only limit their ownexpression but can also affect cellular metabolism). However, packagingof an AAV vector into viral particles still relies on the presence of asuitable helper virus for AAV or the provision of helper virusfunctions. Helper viruses capable of supporting AAV replication areexemplified by adenovirus, but include other viruses such as herpes andpox viruses. The presence of significant quantities of infectious helpervirus in a preparation of AAV vectors is problematic in that thepreparation is intended for use in human administration. Even thepresence of non-replicative helper virus components can cause anunacceptable immunological reaction in the treated subject.

The potential problems elicited by helper virus antigen have beenillustrated in several recent studies. Byrnes et al. (Neuroscience66:1015, 1995) injected an El-region deleted, non-replicating humanadenovirus type 5 into the brains of inbred rats. An inflammatoryresponse was observed that was attributed to the particles administeredrather than to expression of new viral proteins due to viral replicationin the cells. Presence of the virus was associated with an increase inMHC Class I gene expression and a heavy infiltration of macrophages andT cells. McCoy et al. (Human Gene Therapy 6:1553, 1995) instilled thelungs of mice with intact adenovirus, adenovirus with incompletegenomes, or adenovirus inactivated with ultraviolet light. All inducedpulmonary inflammation, and the number of inflammatory cells in the lungtissue was quantitatively similar for all three forms of the virus.Comparative experiments using adenovirus constructs in normal andimmune-deficient mice performed by Barr et al. (Gene Therapy 2:151,1995) indicate that the anti-adenovirus immune response is primarilyT-cell mediated and gives rise to a memory response that affectssubsequent doses.

Accordingly, in the development of recombinant AAV vectors such as thosefor use in gene therapy, there is a need for strategies that minimizethe amount of helper virus, as well as helper virus proteins andcellular proteins, present in the final preparation, while at the sametime still achieving a high titer of AAV so that the methods can beeffectively employed on a scale that is suitable for the practicalapplication of gene therapy techniques.

Since high titers of rAAV vector preparations are particularly useful,but the production of high titers of rAAV, particularly in large-scaleprocedures, can lead to the generation of significant quantities ofcontaminating helper virus (e.g. adenovirus or “Ad”), helper virusproteins (e.g. Ad proteins), and/or cellular proteins, it becameespecially important to design scalable methods for the production ofrAAV that can be used for the generation of high-titer preparations thatare substantially free of contaminating virus and/or viral or cellularproteins.

Prior art methods used to produce recombinant AAV particle usingpackaging cells required a cell lysis step due to the pervasive beliefthat AAV is not released from producer cells in any appreciable amountwithout lysing the cells. See, for example, Chirico and Trempe (1998) J.Viral. Methods 76:3141. However, the cell lysate contains variouscellular components which must be separated from the rAAV vector beforeit is suitable for in vivo use.

The present disclosure provides methods for achieving high-titerproduction of rAAV vectors, including rAAV released from a producer cellwithout lysing the cell(s), and demonstrates that such techniques can beemployed for the large-scale production of recombinant AAV vectorpreparations.

SUMMARY OF THE INVENTION

This invention provides methods and materials for generating high titerpreparations of adeno-associated virus (AAV) that are substantially freeof helper virus, helper virus proteins, and cellular proteins and othercomponents. These methods entail upstream processing (such as growth insuspension and/or under conditions that permit release of virus) as wellas downstream processing (such as chromatography). The upstream anddownstream methods may be used alone or in various combinations.

Accordingly, in one aspect, the invention provides methods of generatinga population of rAAV particles comprising the step of: incubating aproducer cell in a cell culture medium, wherein said producer cell iscultured under suspension conditions, whereby greater than about 10²particles are produced from the producer cell. In some embodiments,tangential flow filtration is employed to purify the population of virusproduced (with or without other steps and conditions as describedherein).

In another aspect, the invention provides methods for generating apopulation of recombinant adeno-associated virus (rAAV) particles,comprising the step of: incubating an AAV producer cell under conditionsthat are permissive for replication of AAV, said producer cellcomprising (i) one or more AAV packaging genes wherein each said AAVpackaging gene encodes an AAV replication or encapsidation protein; (ii)a recombinant AAV (rAAV) vector that comprises a heterologous non-AAVpolynucleotide flanked by at least one AAV inverted terminal repeat(ITR); and (iii) a helper virus for AAV, wherein said helper virus is atemperature-sensitive helper virus, wherein the incubating the producercell line is conducted at a temperature that is permissive forreplication of AAV but non-permissive for replication of thetemperature-sensitive helper virus, whereby AAV virus particles areproduced. In some embodiments, the incubation occurs for at least fivedays from the time of introduction of the temperature-sensitiveadenovirus. In some embodiments, the temperature sensitive helper virusis adenovirus Ad-ts149. The temperature-sensitive helper virus may be inthe form of a virus particle or plasmid. In some embodiments, rAAVproduction is increased at least about 5-fold as compared to rAAVproduction using a wild type adenovirus.

In another aspect, the invention provides methods for isolating apopulation of rAAV particles, comprising the steps of: (a)chromatographing an AAV producer cell lysate containing rAAV particleson a positively-charged anion exchange resin (i.e., at least onepositively-charged anion exchange resin); and (b) chromatographing anAAV producer cell lysate containing rAAV particles on anegatively-charged cation exchange resin (i.e., at least one negativelycharged cation exchange resin), whereby a purified population of rAAVparticles is generated. The steps may be performed in either order. Insome embodiments, an additional step of subjecting the producer cells totangential flow filtration is performed. In some embodiments, anadditional step of subjecting the lysate to tangential flow filtration,which can be before and/or after performing chromatography. Thesemethods are applicable to cells which are adherent or cells which aregrown in suspension.

In another aspect, the invention provides methods for isolating apopulation of rAAV particles, comprising the steps of: (a)chromatographing AAV producer cell culture supernatant which containsrAAV particles on a positively-charged anion exchange resin; and (b)chromatographing the AAV producer cell culture supernatant containingrAAV particles on a negatively-charged cation exchange resin, whereby apurified population of rAAV particles is generated. The steps may beperformed in either order. In some embodiments, an additional step ofsubjecting the supernatant to tangential flow filtration is performed,which may be before and/or after chromatography. These methods areapplicable to cells which are adherent or cells which are grown insuspension.

In another aspect, the invention provides methods for isolating rAAVparticles comprising the steps of (a) chromatographing an AAV producercell lysate containing rAAV particles on a positively charged anionexchange resin; and (b) subjecting the product of step a to tangentialflow filtration to generate a purified population of rAAV. The steps maybe -performed in either order. These methods are applicable to cellswhich are adherent or cells which are grown in suspension.

In another aspect, the invention provides methods for isolating rAAVparticles comprising the steps of (a) chromatographing an AAV producercell culture supernatant which contains rAAV particles on a positivelycharged anion exchange resin; and (b) subjecting the product of step ato tangential flow filtration to generate a purified population of rAAV.The steps may be performed in either order. These methods are applicableto cells which are adherent or cells which are grown in suspension.

In another aspect, the invention provides methods for generating apopulation of rAAV particles comprising culturing a producer cell undera stress condition, said producer cell comprising (i) one or more AAVpackaging genes, wherein each said AAV packaging gene encodes an AAVreplication or encapsidation protein; (ii) a recombinant AAV (rAAV)vector that comprises a heterologous non-AAV polynucleotide flanked byat least one AAV inverted terminal repeat (ITR); and (iii) helper virusfunction for AAV, whereby about two-fold or more rAAV particles areproduced compared to a producer cell not grown under said stresscondition. Examples of stress conditions are provided herein. Thesemethods are applicable to cells which are adherent or cells which aregrown in suspension.

Other embodiments of the invention include but are not limited to thefollowing:

A method of generating a population of recombinant adeno-associatedvirus (rAAV) particles, comprising the steps of: a) providing an AAVproducer cell that comprises: (i) one or more AAV packaging genes,wherein each said AAV packaging gene encodes an AAV replication orencapsidation protein; (ii) a recombinant AAV (rAAV) pro-vector thatcomprises a heterologous non-AAV polynucleotide flanked by at least oneAAV inverted terminal repeat (ITR); and (iii) a helper virus for AAV; b)incubating the producer cell provided in step a) under conditions thatare permissive for replication of AAV; c) lysing the producer cell afterthe incubation of step b) to produce an AAV producer cell lysate; and d)chromatographing the AAV producer cell lysate of step c) on a pluralityof ion-exchange resins comprising at least one positively-charged anionexchange resin and at least one negatively-charged cationic exchangeresin to generate a purified population of rAAV vector particles, orchromatographing the AAV producer cell lysate of step c) on an anionexchange resin followed by tangential flow filtration (TFF).

A method of generating a population of rAAV particles, wherein saidhelper virus is an adenovirus or a temperature-sensitive helper virus,and said step of incubating the producer cell is conducted at atemperature that is permissive for replication of AAV but non-permissivefor replication of the temperature-sensitive helper virus.

A method of generating a population of rAAV particles, whereinincubating the producer cell is conducted in a vessel selected from thegroup consisting of a tissue culture flask, a roller bottle, a spinnerflask, a tank reactor, a fermentor, and a bioreactor, optionally using amicrocarrier, and preferably using a suspension-adapted mammalian cellline.

A method of generating a population of recombinant adeno-associatedvirus (rAAV) particles, comprising the steps of: a) providing an AAVproducer cell that comprises: (i) one or more AAV packaging genes,wherein each said AAV packaging gene encodes an AAV replication orencapsidation protein; (ii) a recombinant AAV (rAAV) pro-vector thatcomprises a heterologous non-AAV polynucleotide flanked by at least oneAAV inverted terminal repeat (ITR); and (iii) a helper virus for AAV ora polynucleotide sequence of said helper virus that encodes at least onehelper virus function; b) subjecting the producer cell provided in stepa) to a sub-lethal stress; and c) incubating the stressed producer cellof step b) under conditions that are permissive for replication of AAV.Possible forms of sub-lethal stress may be selected but are not limitedto those in the group consisting of a nutritional stress, an osmoticstress, a pH stress, a temperature stress, an aerobic stress, amechanical stress, a radiational stress and a toxic stress. Anon-limiting example by which nutritional stress is imposed is byculturing the producer cells in a medium that is deficient in one ormore amino acids. Additional illustrations are provided below.

A method of generating a population of rAAV particles, wherein saidpurified population of rAAV vector particles is substantially free ofreplication-competent AAV and of helper virus and cellular proteins.

A method of generating a population of recombinant adeno-associatedvirus (rAAV) particles, comprising the steps of: a) providing an AAVproducer cell that comprises: (i) one or more AAV packaging genes,wherein each said AAV packaging gene encodes an AAV replication orencapsidation protein; (ii) a recombinant AAV (rAAV) pro-vector thatcomprises a heterologous non-AAV polynucleotide flanked by at least oneAAV inverted terminal repeat (ITR); and (iii) a helper virus for AAV; b)incubating the producer cell provided in step a) under conditions thatare permissive for replication of AAV and which comprise inducing asub-lethal stress in the AAV producer cell; c) lysing the producer cellafter the incubation of step b) to produce an AAV producer cell lysate;and d) purifying the AAV producer cell lysate to generate a populationof recombinant adeno-associated virus (rAAV) particles. Suitablepurification methods include those described elsewhere in thisdisclosure. An exemplary purification procedure compriseschromatographing the AAV producer cell lysate of step c) on at least onechromatographic resin selected from the group consisting of apositively-charged anion exchange resin and a negatively-chargedcationic exchange resin to generate a purified population of rAAV vectorparticles (preferred methods include anion exchange followed by cationexchange or tangential flow filtration (TFF)). Illustrativechromatographic procedures, including ion exchange chromatography, andchromatographic purification on heparin sulfate are provided below byway of example.

A host cell for producing recombinant adeno-associated virus (rAAV)particles at high efficiency, comprising: a) one or more AAV packaginggenes, wherein each said AAV packaging gene encodes an AAV replicationor encapsidation protein; b) a heterologous polynucleotide introducedinto said host cell using an rAAV pro-vector, wherein the rAAVpro-vector comprises the heterologous polynucleotide flanked by at leastone AAV inverted terminal repeat (ITR) and is deficient in said AAVpackaging gene(s); c) a helper virus such as a temperature-sensitivehelper virus (tsHV) for AAV, wherein said tsHV is temperature-sensitivefor self-replication.

In other embodiments, the methods entail release of rAAV particle fromproducer cells without actively lysing the cells as is typical in theart, which provides a distinct and significant advantage over previouslydescribed production methods. The methods are also applicable to viruseswhich are non-lytic and/or generally not released (i.e., non-buddingviruses).

Accordingly, in one aspect, the invention provides methods of generatinga population of virus particles, such as recombinant adeno-associatedvirus (rAAV) particles, comprising the step of: a) incubating a producercell in a cell culture medium under conditions which promote release ofAAV particles from the cell, whereby rAAV particles are released fromthe producer cell into the culture medium, and wherein the producer cellcomprises (i) one or more AAV packaging genes, wherein each said AAVpackaging gene encodes an AAV replication or encapsidation protein; (ii)a recombinant AAV (rAAV) vector that comprises a heterologous non-AAVpolynucleotide flanked by at least one AAV inverted terminal repeat(ITR); and (iii) a helper virus for AAV or helper virus function forAAV. The released rAAV particles may then be collected, or harvested,from the culture medium. Conditions which promote release of rAAV viralparticles are described herein and include, but are not limited to, pH,osmolality, dissolved oxygen, enriched media, and temperature.

In some embodiments, the methods further include various purificationand/or inactivation steps. In some of these embodiments, the methodfurther comprises the steps of: chromatographing the AAV producer cellsupernatant on a plurality of ion-exchange resins comprising at leastone positively-charged anion exchange resin and at least onenegatively-charged cationic exchange resin to generate a purifiedpopulation of rAAV vector particles, or chromatographing the AAVproducer cell supernatant on an anion exchange resin followed bytangential flow filtration (TFF). Heparin sulphate chromotography canalso be used as a cation exchange resin to further purify the virus.

In the methods of the invention, cell culture can be carried out suchthat the cells are in suspension or under conditions that promoteadherence of cells to a solid support. Accordingly, in some embodiments,the producer cell is cultured in a vessel selected from the groupconsisting of a tissue culture flask, a roller bottle, a spinner flask,a tank reactor, a fermentor, and a bioreactor, a flat stock reactor, ahollow fiber system, a packed bed reactor and optionally using amicrocarrier.

In some embodiments of the invention, recombinant AAV vectorpreparations produced by the methods result in a purified population ofrAAV vector particles which is substantially free ofreplication-competent AAV and of helper virus and cellular proteins aswell as substantially free cellular DNA.

The present invention further provides a population of rAAV particles,produced according to any of the production methods of this inventionPreferably, the population of particles contains no more than about oneinfectious adenovirus particles per thousand infectious rAAV particles,preferably less than one per 10⁶ rAAV, still more preferably less thanabout one in 10⁹, even more preferably less than about one in 10¹⁰.

Also provided are high-throughput assay techniques which can be used,for example, in the titering of virus preparations as well as in thescreening of agents that affect viral infectivity and/or replication.

These and other embodiments of the invention are outlined in thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a half-tone reproduction of a Southern analysis for rAAVvector production, using a probe for a model CF therapeutic genecontained in the vector. The prominent band at 1.4 kb indicates presenceof rAAV in the preparation. Helper function was supplied by adenovirussubtype 5 (Ad5) or by the adenovirus temperature-sensitive strain ts149.

FIG. 2 is a half-tone reproduction of a slot-blot analysis for rAAVvector production, to quantitate the level of rAAV present in eachpreparation. When helper function is supplied by ts149, the amount ofrAAV produced under standard culture conditions is several logs belowthat produced in the presence of Ad5.

FIG. 3 is a half-tone reproduction of a Southern analysis for rAAV,indicating that increasing the level of ts149 does not improve the levelof rAAV production.

FIG. 4 is a bar graph indicating a dramatic increase in the amount ofrAAV produced in the presence of ts149 (hatched bars) if culture periodsare extended beyond 5 days. This is in marked contrast to thesubstantial decrease in rAAV that occurs beyond day 5 whennon-temperature sensitive adenovirus is used to supply helper function(solid bars).

FIG. 5 is a line graph showing the viable cell density (VCD) of HeLa S3cells grown in suspension culture at 37° C. (circles) or 32° C.(squares).

FIG. 6 is a line graph showing the effect of tangential flow filtrationat two different rates on HeLa S3 cells grown in suspension culture.

FIG. 7 is a bar graph showing the production of ts149 detected ininfected HeLa S3 cells cultured for 3-7 days in suspension at thepermissive temperature of 32° C., compared with the level detected atday 7 after microfluidization (MF).

FIG. 8 is a combination graph showing the purification of ts149 by anionexchange chromatography on PI matrix, eluted with a linear 900-1300 meqNaCl gradient at pH 8.0.

FIG. 9 is a combination graph showing the purification of Adenovirus onPI anion-exchange matrix, eluted with a 800-1300 meq gradient of NaCl atpH8.0. Bars: Viral activity measured in an infectivity assay; Solidline: A₂₈₀ (a measure of total protein); Dotted line: bufferconductivity (mS).

FIG. 10 is a combination graph showing the separation of Adenovirus andrecombinant AAV. The upper panel shows separation on PI anion-exchangematrix, eluted with a 0-1000 meq gradient of NaCl at pH 8.0. The lowerpanel shows subsequent separation of Adenovirus from contaminants on HScation-exchange matrix, eluted with a 0-500 meq gradient of NaCl at pH8.0.

FIG. 11 is two bar graphs, showing the effect of fetal bovine serumlevels (FBS) in the culture medium on rAAV production. The upper graphindicates DRPs; the lower graph indicates RUs. Serum deficiency in theculture medium is one of a number of stress factors that the producercells can be subjected to in order to enhance the production of viralparticles.

FIG. 12 is a half-tone reproduction of a SDS-polyacrylamide gel analysisfor AAV proteins during purification steps. The AAV preparation wassubjected to tangential flow filtration after chromatography on an anionexchange column (POROS 50 PI). The silver stained gel shows the highlypurified AAV capsid proteins, VP1, VP2, and VP3 in the final bulkmaterial.

FIG. 13 is a chromatogram showing concentration of AAV on a heparinsulfate column. The sharp peak in absorbance at 280 nm (left-hand axis)at about 18 minutes elution time represents the AAV fraction (afteranion exchange and tangential flow filtration) as eluted from heparinsulfate with a linear gradient of 0 to 1M NaCl (conductivity in ms shownon right-hand axis).

FIGS. 14A and 14B are bar graphs depicting the results of two separateexperiments, expressed as DNase resistant particles (DRP) per cell atthe various pH levels. Cell cultures were maintained at the indicated pHlevels, and cell lysates were assayed at day 2 (solid bars) and day 3(hatched bars) post-infection.

FIGS. 15A and 15B are bar graphs and depict the results, expressed astotal DRPs, of rAAV production, at day 2 (FIG. 15A) and day 3 (FIG. 15B)post-infection, in bioreactors maintained at various pH levels.Percentages above each bar are percentages of total DRPs in the celllysate. The solid portion of each bar represents DRPs in cell lysates,while the hatched portion of each bar represents the DRPs in the cellculture medium. Percentages above each bar indicate the percentage oftotal DRPs in the cell lysate.

FIGS. 16A and 16B are bar graphs depicting the total replication units(RU), at day 2 (FIG. 16A) and day 3 (FIG. 16B) post-infection, in theculture media (hatched portion of each bar) and cell lysates (solidportion of each bar) when cultures were maintained at the indicated pHlevels. Percentages above each bar indicate the percentage of total RUsin the cell lysate.

FIG. 17 is a bar graph depicting the particle:infectivity (P/I) ratio ofrAAV particles harvested from cell lysates (solid portion of each bar)and cell culture medium (hatched portion of each bar) at day 3post-infection from bioreactors maintained at the indicated pH levels.

FIGS. 18A, 18B, and 18C are bar graphs depicting the total DRPs in celllysates (solid portion of each bar) and cell culture media (hatchedportion of each bar) on day 2 (FIG. 18A), day 3 (FIG. 18B), and day 4(FIG. 18C) post-infection in bioreactors in which the cell culture mediacontained the indicated starting osmolality. Percentages above each barindicate the percentage of total DRPs in the cell lysate.

FIGS. 19A, 19B, and 19C are bar graphs depicting the total RUs in celllysates (solid portion of each bar) and cell culture media (hatchedportion of each bar) on day 2 (FIG. 19A), day 3 (FIG. 19B), and day 4(FIG. 19C) post-infection in bioreactors in which the cell culture mediacontained the indicated starting osmolality. Percentages above each barindicate the percentage of total RUs in the cell lysates.

FIG. 20 is a bar graph depicting the P/I ratio of rAAV particles in cellculture media at days 3 and 4 from bioreactor cultures with theindicated starting osmolalities.

FIGS. 21 A-C are bar graphs depicting the total DRPs in cell lysates(solid portion of each bar) and cell culture media (hatched portion ofeach bar) on day 2 (FIG. 21A), day 3 (FIG. 21B), and day 4 (FIG. 21C)post-infection in bioreactors in which the cell culture media wasmaintained at the indicated temperature. Percentages above each barindicate the percentage of total DRPs in the cell lysate.

FIGS. 22A-C are bar graphs depicting the total RUs in cell lysates(solid portion of each bar) and cell culture media (hatched portion ofeach bar) on day 2 (FIG. 22A), day 3 (FIG. 22B), and day 4 (FIG. 22C)post-infection in bioreactors in which the cell culture media wasmaintained at the indicated temperature. Percentages above each bar inFIG. 22A indicate the percentage of total RUs in the cell lysate.

FIG. 23 is a bar graph depicting the total DRPs in the culture mediathree days post-infection in cultures grown in the various mediaindicated.

FIG. 24 is a bar graph depicting the RUs in the culture media three dayspost-infection in cultures grown in the various media indicated

FIG. 25 is a bar graph depicting the P/I ratio of viral particles in thecell culture media when cultures were grown in the various mediaindicated.

FIG. 26 provides amino acid and vitamin compositions for the mediasupplements described in Example 17.

FIG. 27 is a bar graph depicting the total cell density at the time ofharvest for the various media formulations.

FIG. 28 is a bar graph depicting the percent of DRPs released in thecell culture medium (white bars) versus those retained in the cell(black bars) from attached cell cultures for the various formulationstested.

FIG. 29 is a bar graph depicting the percent of RUs released in the cellculture medium (white bars) versus those retained in the cell (blackbars) from attached cell cultures for the various formulations tested.

FIG. 30 is a bar graph depicting the particle infectivity (P/I) ratio ofrAAV particles harvested from cell lysates (white bars) and cell culturemedium (black bars) from attached cell cultures for the various mediaformulations.

FIGS. 31A and 31B are bar graphs depicting the total DRPs (FIG. 31A) andRUs (FIG. 31B) in cell lysates (solid portion of each bar) and cellculture media (hatched portion of each bar) for attached cell culturesadjusted to 450 mOsm with NaCl at the times indicated in Table 9.

FIG. 32 is a bar depicting the particle infectivity (P/I) ratio of rAAVparticles harvested from cell lysates (black bars) and cell culturemedium (hatched bars) from attached cell cultures adjusted to 450 mOsmwith NaCl at the times indicated in Table 9.

FIGS. 33A and 33B are graphs depicting total cell density for NaCl (FIG.33A) and sorbitol (FIG. 33B) formulated cultures of various osmolalitiesand conductivities over time with rAAV vector production.

FIGS. 34A and 34B are graphs depicting glucose consumption rates ofcells in NaCl (FIG. 34A) and sorbitol (FIG. 34B) formulated cultures ofvarious osmolalities and conductivities over time with rAAV vectorproduction.

FIGS. 35A-D are bar graphs depicting the total DRPs in cell lysates(solid portion of each bar) and cell culture media (hatched portion ofeach bar) on day 2 (FIGS. 35A and 35C) and day 3 (FIGS. 35B and 35D)post infection in bioreactors for media formulated at the indicatedstarting osmolality with NaCl (FIGS. 35 A and 35B) or sorbitolrespectively. Percentages above each bar indicate the total DRPs in thecell lysate. The sorbitol control in FIGS. 35A and 35B represent thebioreactor formulated at 300 mOsm with sorbitol; the NaCl control inFIGS. 35C and 35D represent the bioreactor formulated with 300 mOsmNaCl.

FIGS. 36A-D are bar graphs depicting the RUs per cell in cell lysates(solid portion of each bar) and cell culture media (hatched portion ofeach bar) on day 2 (FIGS. 36A and 36C) and day 3 (FIGS. 36B and 36D)post infection in bioreactors for media formulated at the indicatedstarting osmolality with NaCl (FIGS. 36A and 36B) or sorbitol (FIGS. 36C and 36D). Percentages above each bar indicate the percent of RUs percell contained in the cell lysate.

FIGS. 37A-D are bar graphs depicting P/I ratios of rAAV particles incell lysates (solid bars) and cell culture media (hatched bars) on day 2(FIGS. 37A and 37C) and day 3 (FIGS. 37B and 37D) post infection inbioreactors for media formulated at the indicated starting osmolalitywith NaCl (FIGS. 37A and 37B) or sorbitol (FIGS. 37C and D).

MODES FOR CARRYING OUT THE INVENTION

It is an object of this invention to provide methods and materials forgenerating high titer preparations of adeno-associated virus (AAV) thatare substantially free of helper virus, helper virus proteins, andcellular proteins and other components.

Various methods for the generation and processing of AAV particles inmammalian cells are described in detail below, and illustrations of theuse of such techniques are provided in the Examples following.

By way of introduction, it is typical to employ a host or “producer”cell for rAAV vector replication and packaging. Such a producer cell(usually a mammalian host cell) generally comprises or is modified tocomprise several different types of components for rAAV production. Thefirst component is a recombinant adeno-associated viral (rAAV) vectorgenome (or “rAAV pro-vector”) that can be replicated and packaged intovector particles by the host packaging cell. The rAAV pro-vector willnormally comprise a heterologous polynucleotide (or “transgene”), withwhich it is desired to genetically alter another cell in the context ofgene therapy (since the packaging of such a transgene into rAAV vectorparticles can be effectively used to deliver the transgene to a varietyof mammalian cells). The transgene is generally flanked by two AAVinverted terminal repeats (ITRs) which comprise sequences that arerecognized during excision, replication and packaging of the AAV vector,as well as during integration of the vector into a host cell genome. Asecond component is a helper virus that can provide helper functions forAAV replication. Although adenovirus is commonly employed, other helperviruses can also be used as is known in the art. Alternatively, therequisite helper virus functions can be isolated genetically from ahelper virus and the encoding genes can be used to provide helper virusfunctions in trans. The AAV vector elements and the helper virus (orhelper virus functions) can be introduced into the host cell eithersimultaneously or sequentially in any order. The final components forAAV production to be provided in the producer cell are “AAV packaginggenes” such as AAV rep and cap genes that provide replication andencapsidation proteins, respectively. Several different versions of AAVpackaging genes can be provided (including wild-type rep-cap cassettesas well as modified rep and/or cap cassettes in which the rep and/or capgenes can be left under the control of the native promoters or operablylinked to heterologous promoters. Such AAV packaging genes can beintroduced either transiently or stably into the host packaging cell, asis known in the art and described in more detail below.

After culturing the host cells under conditions that permit AAVreplication and encapsidation, the cells and sub-cellular fractions canbe processed to generate high titer preparations of adeno-associatedvirus (AAV) that are substantially free of helper virus, helper virusproteins, and cellular proteins. Detailed descriptions of processingtechniques and illustrative protocols employing such techniques areprovided below.

In some embodiments, the methods generally entail culturing (whichgenerally involves maintaining) producer cells under conditions whichpromote release of rAAV particles from the producer cells. Followingthese methods of the invention, rAAV particles are released into thecell culture medium (“supernatant”) from intact (i.e., not lysed) cells.After culturing the host cells under conditions that permit AAVreplication, encapsidation, and release the supernatant can be processedto generate high titer preparations of adeno-associated virus (AAV) thatare substantially free of helper virus, helper virus proteins, cellularproteins, and, significantly, cellular DNA. Detailed descriptions ofprocessing techniques and illustrative protocols employing suchtechniques are provided below.

It is well-established in the AAV field that AAV is not released fromthe cell unless the cell is lysed, but remains in the nucleus of thecell. Accordingly, the pervasive and universal belief is that, in orderto produce rAAV particles, the cells must be lysed. In contrast to theteachings of the field, we have discovered that rAAV particles can bereleased from cells without lysing the cells, and further that releaseof rAAV particles can be increased by maintaining the producer cellsunder various controlled environmental conditions. Using theseconditions, rAAV particles can be produced at titers higher thanpreviously obtained. Cells cultured under the conditions describedherein produce more virus per cell and release more virus into theculture medium, and, even more significantly, may release a populationof AAV with higher infectivity than AAV which is retained within thecell. In other words, the DNAse resistant particle to infectivity ratiocan be smaller in the AAV population released into the cell culturemedium compared to this ratio of AAV retained within the cell (see,e.g., FIG. 4). Furthermore, since lysis is not an obligatory step in themethods of the present invention, the rAAV particles can be collectedfrom the cell supernatant, thus simplifying subsequent optionalpurification steps. Alternatively, lysis could also be performed.

In some embodiments, the invention provides methods of release, orpreferential release, of infectious viral particles. This preferentialrelease of infectious particles is particularly significant in the viralproduction context, in which it is highly desirable to produce apopulation containing a large number of infectious particles as opposedto noninfective particles.

It is understood that the methods and principles described herein areapplicable to a number of other viruses which are normally retained(i.e., not released), particularly adenovirus. AAV is exemplifiedherein.

The rAAV particles produced by the methods of this invention areparticularly useful as gene transfer vectors. Methods of using suchvectors are known in the art and need not be described herein.

Definitions

A “vector” as used herein refers to a macromolecule or association ofmacromolecules that comprises or associates with a polynucleotide andwhich can be used to mediate delivery of the polynucleotide to a cell.Illustrative vectors include, for example, plasmids, viral vectors,liposomes and other gene delivery vehicles.

“AAV” is an abbreviation for adeno-associated virus, and may be used torefer to the virus itself or derivatives thereof. The term covers allsubtypes and both naturally occurring and recombinant forms, exceptwhere required otherwise. The abbreviation “rAAV” refers to recombinantadeno-associated virus, also referred to as a recombinant AAV vector (or“rAAV vector”).

An “rAAV vector” as used herein refers to an AAV vector comprising apolynucleotide sequence not of AAV origin (i.e., a polynucleotideheterologous to AAV), typically a sequence of interest for the genetictransformation of a cell. In preferred vector constructs of thisinvention, the heterologous polynucleotide is flanked by at least one,preferably two AAV inverted terminal repeat sequences (ITRs). The termrAAV vector encompasses both rAAV vector particles and rAAV vectorplasmids.

An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refersto a viral particle composed of at least one AAV capsid protein(preferably by all of the capsid proteins of a wild-type AAV) and anencapsidated polynucleotide rAAV vector. If the particle comprises aheterologous polynucleotide (i.e. a polynucleotide other than awild-type AAV genome such as a transgene to be delivered to a mammaliancell), it is typically referred to as an “rAAV vector particle” orsimply an “rAAV vector”. Thus, production of rAAV particle necessarilyincludes production of rAAV vector, as such a vector is contained withinan rAAV particle.

“Packaging” refers to a series of intracellular events that result inthe assembly and encapsidation of an AAV particle.

AAV “rep” and “cap” genes refer to polynucleotide sequences encodingreplication and encapsidation proteins of adeno-associated virus. Theyhave been found in all AAV serotypes examined, and are described belowand in the art. AAV rep and cap are referred to herein as AAV “packaginggenes”.

A “helper virus” for AAV refers to a virus that allows AAV (e.g.wild-type AAV) to be replicated and packaged by a mammalian cell. Avariety of such helper viruses for AAV are known in the art, includingadenoviruses, herpesviruses and poxviruses such as vaccinia. Theadenoviruses encompass a number of different subgroups, althoughAdenovirus type 5 of subgroup C is most commonly used. Numerousadenoviruses of human, non-human mammalian and avian origin are knownand available from depositories such as the ATCC. Viruses of the herpesfamily include, for example, herpes simplex viruses (HSV) andEpstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) andpseudorabies viruses (PRV); which are also available from depositoriessuch as ATCC.

“Helper virus function(s)”, refers to function(s) encoded in a helpervirus genome which allow AAV replication and packaging (in conjunctionwith other requirements for replication and packaging described herein).As described herein, “helper virus function” may be provided in a numberof ways, including by providing helper virus or providing, for example,polynucleotide sequences encoding the requiste function(s) to a producercell in trans.

The term “tsHV” refers to a temperature-sensitive helper virus, whichcan provide helper functions for AAV replication and packaging but istemperature-sensitive with respect to its own replication (i.e. it canreplicate at a “permissive” temperature but replicates at lowerefficiency, or preferably not at all, at a “non-permissive”temperature). The ability of the tsHV to provide help for AAVreplication may also be temperature sensitive, but preferred tsHV foruse with this invention efficiently support AAV replication attemperatures at which AAV can replicate but which are non-permissive forreplication of the tsHV. Examples of such tsHV are described below.

An “infectious” virus or viral particle is one that comprises apolynucleotide component which it is capable of delivering into a cellfor which the viral species is trophic. The term does not necessarilyimply any replication capacity of the virus. Assays for countinginfectious viral particles are described elsewhere in this disclosureand in the art. Viral infectivity can be expressed as the P:I ratio, orthe ratio of total viral particles to infective viral particles.

A “replication-competent” virus (e.g. a replication-competent AAV)refers to a phenotypically wild-type virus that is infectious, and isalso capable of being replicated in an infected cell (i.e. in thepresence of a helper virus or helper virus functions). In the case ofAAV, replication competence generally requires the presence offunctional AAV packaging genes. Preferred rAAV vectors as describedherein are replication-incompetent in mammalian cells (especially inhuman cells) by virtue of the lack of one or more AAV packaging genes.Preferably, such rAAV vectors lack any AAV packaging gene sequences inorder to minimize the possibility that replication competent AAV aregenerated by recombination between AAV packaging genes and an incomingrAAV vector. Preferred rAAV vector preparations as described herein arethose which contain few if any replication competent AAV (rcAAV, alsoreferred to as RCA) (preferably less than about 1 rcAAV per 10² rAAVparticles, more preferably less than about 1 rcAAV per 10⁴ rAAVparticles, still more preferably less than about 1 rcAAV per 10⁸ rAAVparticles, even more preferably less than about 1 rcAAV per 10¹² rAAVparticles, most preferably no rcAAV).

“Release” of rAAV particles means that rAAV particles enter the cellculture medium from an intact producer cell, i.e., the rAAV particle isreleased without lysing the cell. It is understood that, in a givenproducer cell culture, some cells lyse, for example, upon cell death.However, this invention provides methods which promote release of rAAVparticle without performing deliberate cell lysis, as it typically donein the art. The terms “release” and “secretion” from a producer cell areused interchangeably herein. As the data disclosed herein indicate,release of rAAV under conditions described herein to promote release isnot due to, for example, lytic function of helper virus.

The term “condition that promotes release of rAAV particles” from aproducer cell, as used herein, refers to a condition for growingproducer cells which lead to increased, or enhanced, rAAV particlerelease from the producer cell into the culture medium. Conditions whichpromote release of rAAV from the producer cell into the culture mediumare described herein, and are generally, but not necessarily, conditionswhich enhance cellular metabolism. “Promoting release” of rAAV particlesfrom a producer cell into the culture medium means that the rAAV releasefrom the producer cell is increased when compared to rAAV release from aproducer cell not cultured under the environmental condition(s) whichenhance release. The increase may be any detectable increase, such as atleast about 1%, at least about 5%, at least about 10%, more preferablyat least about 20%, more preferably at least about 25%, more preferablyat least about 35%, more preferably at least about 50%, more preferablyat least about 60%, more preferably at least about 65%, more preferablyat least about 75%, more preferably at least about 80%, more preferablyat least about 85%, more preferably at least about 90%, more preferablyat least about 100% or 2-fold, more preferably at least about 5-fold,more preferably at least about 10-fold, more preferably at least about20-fold, even more preferably at least about 50-fold. As is well knownin the art, when a cell population is grown under a given startingculture condition, the cells' metabolic by-products will change certainof the culture conditions, such as pH and osmolality. Underenvironmental conditions which promote rAAV particle release, one ormore of these parameters is controlled as necessary, i.e, monitored atregular intervals and adjusted to maintain the parameter within asuitable range. (i.e., a range that promotes release). Setting and/orcontrol of these conditions will be discussed in more detail below.

The term “polynucleotide” refers to a polymeric form of nucleotides ofany length, including deoxyribonucleotides or ribonucleotides, oranalogs thereof. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs, and may beinterrupted by non-nucleotide components. If present, modifications tothe nucleotide structure may be imparted before or after assembly of thepolymer. The term polynucleotide, as used herein, refers interchangeablyto double- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment of the invention described herein that is apolynucleotide encompasses both the double-stranded form and each of twocomplementary single-stranded forms known or predicted to make up thedouble-stranded form.

A “gene” refers to a polynucleotide containing at least one open readingframe that is capable of encoding a particular protein after beingtranscribed and translated.

“Recombinant”, as applied to a polynucleotide means that thepolynucleotide is the product of various combinations of cloning,restriction or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature. Arecombinant virus is a viral particle comprising a recombinantpolynucleotide. The terms respectively include replicates of theoriginal polynucleotide construct and progeny of the original virusconstruct.

A “control element” or “control sequence” is a nucleotide sequenceinvolved in an interaction of molecules that contributes to thefunctional regulation of a polynucleotide, including replication,duplication, transcription, splicing, translation, or degradation of thepolynucleotide. The regulation may affect the frequency, speed, orspecificity of the process, and may be enhancing or inhibitory innature. Control elements known in the art include, for example,transcriptional regulatory sequences such as promoters and enhancers. Apromoter is a DNA region capable under certain conditions of binding RNApolymerase and initiating transcription of a coding region usuallylocated downstream (in the 3′ direction) from the promoter.

“Operatively linked” or “operably linked” refers to a juxtaposition ofgenetic elements, wherein the elements are in a relationship permittingthem to operate in the expected manner. For instance, a promoter isoperatively linked to a coding region if the promoter helps initiatetranscription of the coding sequence. There may be intervening residuesbetween the promoter and coding region so long as this functionalrelationship is maintained.

An “expression vector” is a vector comprising a region which encodes apolypeptide of interest, and is used for effecting the expression of theprotein in an intended target cell. An expression vector also comprisescontrol elements operatively linked to the encoding region to facilitateexpression of the protein in the target. The combination of controlelements and a gene or genes to which they are operably linked forexpression is sometimes referred to as an “expression cassette,” a largenumber of which are known and available in the art or can be readilyconstructed from components that are available in the art.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is being compared. Forexample, a polynucleotide introduced by genetic engineering techniquesinto a plasmid or vector derived from a different species is aheterologous polynucleotide. A promoter removed from its native codingsequence and operatively linked to a coding sequence with which it isnot naturally found linked is a heterologous promoter.

“Genetic alteration” refers to a process wherein a genetic element isintroduced into a cell other than by mitosis or meiosis. The element maybe heterologous to the cell, or it may be an additional copy or improvedversion of an element already present in the cell. Genetic alterationmay be effected, for example, by transfecting a cell with a recombinantplasmid or other polynucleotide through any process known in the art,such as electroporation, calcium phosphate precipitation, or contactingwith a polynucleotide-liposome complex. Genetic alteration may also beeffected, for example, by transduction or infection with a DNA or RNAvirus or viral vector. Preferably, the genetic element is introducedinto a chromosome or mini-chromosome in the cell; but any alterationthat changes the phenotype and/or genotype of the cell and its progenyis included in this term.

A cell is said to be “stably” altered, transduced, or transformed with agenetic sequence if the sequence is available to perform its functionduring extended culture of the cell in vitro. In preferred examples,such a cell is “inheritably” altered in that a genetic alteration isintroduced which is also inheritable by progeny of the altered cell.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The terms also encompass an amino acid polymer that has beenmodified; for example, disulfide bond formation, glycosylation,lipidation, or conjugation with a labeling component. Polypeptides suchas “CFTR”, “p53”, “E1A” and the like, when discussed in the context ofgene therapy and compositions therefor, refer to the respective intactpolypeptide, or any fragment or genetically engineered derivativethereof, that retains the desired biochemical function of the intactprotein. Similarly, references to CFTR, p53, E1A genes, and other suchgenes for use in gene therapy (typically referred to as “transgenes” tobe delivered to a recipient cell), include polynucleotides encoding theintact polypeptide or any fragment or genetically engineered derivativepossessing the desired biochemical function.

An “isolated” plasmid, virus, or other substance refers to a preparationof the substance devoid of at least some of the other components thatmay also be present where the substance or a similar substance naturallyoccurs or is initially prepared from. Thus, for example, an isolatedsubstance may be prepared by using a purification technique to enrich itfrom a source mixture. Enrichment can be measured on an absolute basis,such as weight per volume of solution, or it can be measured in relationto a second, potentially interfering substance present in the sourcemixture. Increasing enrichments of the embodiments of this invention areincreasingly more preferred. Thus, for example, a 2-fold enrichment ispreferred, 10-fold enrichment is more preferred, 100-fold enrichment ismore preferred, 1000-fold enrichment is even more preferred.

A preparation of AAV is said to be “substantially free” of helper virusif the ratio of infectious AAV particles to infectious helper virusparticles is at least about 10²:1; preferably at least about 10⁴:1, morepreferably at least about 10⁶:1; still more preferably at least about10⁸:1. Preparations are also preferably free of equivalent amountsof-helper virus proteins (i.e. proteins as would be present as a resultof such a level of helper virus if the helper virus particle impuritiesnoted above were present in disrupted form). Viral and/or cellularprotein contamination can generally be observed as the presence ofCoomassie staining bands or silver stained bands on SDS gels (e.g. theappearance of bands other than those corresponding to the AAV capsidproteins VP1, VP2 and VP3).

“Efficiency” when used in describing viral production, replication orpackaging refers to useful properties of the method; in particular, thegrowth rate and the number of virus particles produced per cell. “Highefficiency” production indicates production of at least 100 viralparticles per cell; preferably at least about 10,000 and more preferablyat least about 100,000 particles per cell, over the course of theculture period specified. Even more preferably, “high efficiency”production encompasses these production levels of particles per cell aswell as the maximum number of cells producing particles, such as atleast about 10%, preferably at least about 20%, preferably at least 30%,preferably at least 50%, preferably at least 75%. Example 6 describesculture conditions (“complete” medium) which resulted in 93,000-123,000rAAV particles per producer cell. In the context of the presentinvention, efficiency may also be considered in terms of percentage, orextent, of release of viral particles compared to viral particlesretained in the cell. Efficiency may also be considered in terms ofratio or relative proportion of total viral particles to infectiousviral particles (such as a “P/I” ratio). Assays for determiningparameters of efficiency of production, such as replicative units andinfectious center assay, are known in the art.

An “individual” or “subject” treated in accordance with this inventionrefers to vertebrates, particularly members of a mammalian species, andincludes but is not limited to domestic animals, sports animals, andprimates, including humans.

“Treatment” of an individual or a cell is any type of intervention in anattempt to alter the natural course of the individual or cell at thetime the treatment is initiated. For example, treatment of an individualmay be undertaken to decrease or limit the pathology caused by anypathological condition, including (but not limited to) an inherited orinduced genetic deficiency, infection by a viral, bacterial, orparasitic organism, a neoplastic or aplastic condition, or an immunesystem dysfunction such as autoimmunity or immunosuppression. Treatmentincludes (but is not limited to) administration of a composition, suchas a pharmaceutical composition, and administration of compatible cellsthat have been treated with a composition. Treatment may be performedeither prophylactically or therapeutically; that is, either prior orsubsequent to the initiation of a pathologic event or contact with anetiologic agent.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, virology,animal cell culture and biochemistry which are within the skill of theart. Such techniques are explained fully in the literature. See, forexample, “Molecular Cloning: A Laboratory Manual”, Second Edition(Sambrook, Fritsch & Maniatis, 1989); “Animal Cell Culture” (R. I.Freshney, ed., 1987); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Calos, eds., 1987); “Current Protocols in MolecularBiology” (F. M. Ausubel et al., eds., 1987); “Current Protocols inProtein Science” (John E Coligan, et al. eds. Wiley and Sons, 1995); and“Protein Purification: Principles and Practice” (Robert K. Scopes,Springer-Verlag, 1994).

All patents, patent applications, articles and publications mentionedherein, both supra and infra, are hereby incorporated herein byreference.

Selection and Preparation of AAV Vector and AAV Packaging Genes

A recombinant AAV vector of this invention comprises a heterologous(i.e. non-AAV) polynucleotide of interest in place of all or a portionof the AAV rep and/or cap genes that normally make up the bulk of theAAV genome. As in the wild-type AAV genome, however, the rAAV pro-vectoris preferably flanked by two AAV inverted terminal repeats (ITRs) asnoted above. Variations in which an rAAV construct is flanked by a onlya single (typically modified) ITR have also been described in the artand can be employed in connection with the present invention.

Adeno-associated viruses of any serotype are suitable, since the variousserotypes are functionally and structurally related, even at the geneticlevel (see, e.g., Blacklow, pp. 165-174 of “Parvoviruses and HumanDisease” J. R. Pattison, ed. (1988); and Rose, Comprehensive Virology3:1, 1974). All AAV serotypes apparently exhibit similar replicationproperties mediated by homologous rep genes; and all generally bearthree related capsid proteins such as those expressed in AAV2. Thedegree of relatedness is further suggested by heteroduplex analysiswhich reveals extensive cross-hybridization between serotypes along thelength of the genome; and the presence of analogous self-annealingsegments at the termini that correspond to ITRS. The similar infectivitypatterns also suggest that the replication functions in each serotypeare under similar regulatory control. Among the various AAV serotypes,AAV2 is most commonly employed.

An AAV vector of this invention will typically comprise a polynucleotidethat is heterologous to the AAV. The polynucleotide is typically ofinterest because of a capacity to provide a function to a target cell inthe context of gene therapy, such as up- or down-regulation of theexpression of a certain phenotype. Such a heterologous polynucleotide or“transgene”, will generally be of sufficient length to provide thedesired function or encoding sequence. For encapisdation within AAV2particles, the transgene will preferably be less than about 5 kbalthough other serotypes and/or modifications may be employed to allowlarger sequences to packaged into the AAV viral particles.

Where transcription of the heterologous polynucleotide is desired in theintended target cell, it can be operably linked to its own or to aheterologous promoter, depending for example on the desired level and/orspecificity of transcription within the target cell, as is known in theart. Various types of promoters and enhancers are suitable for use inthis context. Constitutive promoters provide an ongoing level of genetranscription, and are preferred when it is desired that the therapeuticpolynucleotide be expressed on an ongoing basis. Inducible promotersgenerally exhibit low activity in the absence of the inducer, and areup-regulated in the presence of the inducer. They may be preferred whenexpression is desired only at certain times or at certain locations, orwhen it is desirable to titrate the level of expression using aninducing agent. Promoters and enhancers may also be tissue-specific:that is, they exhibit their activity only in certain cell types,presumably due to gene regulatory elements found uniquely in thosecells.

Illustrative examples of promoters are the SV40 late promoter fromsimian virus 40, the Baculovirus polyhedron enhancer/promoter element,Herpes Simplex Virus thymidine kinase (HSV tk), the immediate earlypromoter from cytomegalovirus (CMV) and various retroviral promotersincluding LTR elements. Inducible promoters include heavy metal ioninducible promoters (such as the mouse mammary tumor virus (mMTV)promoter or various growth hormone promoters), and the promoters from T7phage which are active in the presence of T7 RNA polymerase. By way ofillustration, examples of tissue-specific promoters include varioussurfactin promoters (for expression in the lung), myosin promoters (forexpression in muscle), and albumin promoters (for expression in theliver). A large variety of other promoters are known and generallyavailable in the art, and the sequences for many such promoters areavailable in sequence databases such as the GenBank database.

Where translation is also desired in the intended target cell, theheterologous polynucleotide will preferably also comprise controlelements that facilitate translation (such as ribosome binding site or“RBS” and a polyadenylation signal). Accordingly, the heterologouspolynucleotide will generally comprise at least one coding regionoperatively linked to a suitable promoter, and may also comprise, forexample, an operatively linked enhancer, ribosome binding site andpoly-A signal. The heterologous polynucleotide may comprise one encodingregion, or more than one encoding regions under the control of the sameor different promoters. The entire unit, containing a combination ofcontrol elements and encoding region, is often referred to as anexpression cassette.

The heterologous polynucleotide is integrated by recombinant techniquesinto or preferably in place of the AAV genomic coding region (i.e. inplace of the AAV rep and cap genes), but is generally flanked on eitherside by AAV inverted terminal repeat (ITR) regions. This means that anITR appears both upstream and downstream from the coding sequence,either in direct juxtaposition, preferably (although not necessarily)without any intervening sequence of AAV origin in order to reduce thelikelihood of recombination that might regenerate areplication-competent AAV genome. Recent evidence suggests that a singleITR can be sufficient to carry out the functions normally associatedwith configurations comprising two ITRs (WO 94/13788), and vectorconstructs with only one ITR can thus be employed in conjunction withthe packaging and production methods of the present invention.

The native promoters for rep are self-regulating, and can limit theamount of AAV particles produced. The rep gene can also be operablylinked to a heterologous promoter, whether rep is provided as part ofthe vector construct, or separately. Any heterologous promoter that isnot strongly down-regulated by rep gene expression is suitable; butinducible promoters are preferred because constitutive expression of therep gene can have a negative impact on the host cell. A large variety ofinducible promoters are known in the art; including, by way ofillustration, heavy metal ion inducible promoters (such asmetallothionein promoters); steroid hormone inducible promoters (such asthe MMTV promoter or growth hormone promoters); and promoters such asthose from T7 phage which are active in the presence of 17 RNApolymerase. An especially preferred sub-class of inducible promoters arethose that are induced by the helper virus that is used to complementthe replication and packaging of the rAAV vector. A number ofhelper-virus-inducible promoters have also been described, including theadenovirus early gene promoter which is inducible by adenovirus E1Aprotein; the adenovirus major late promoter; the herpesvirus promoterwhich is inducible by herpesvirus proteins such as VP16 or 1CP4; as wellas vaccinia or poxvirus inducible promoters.

Methods for identifying and testing helper-virus-inducible promotershave been described in a commonly-owned copending application publishedas WO96/17947 by Targeted Genetics Corporation (Allen et al.). Thus,methods are known in the art to determine whether or not candidatepromoters are helper-virus-inducible, and whether or not they will beuseful in the generation of high efficiency packaging cells. Briefly,one such method involves replacing the p5 promoter of the AAV rep genewith the putative helper-virus-inducible promoter (either known in theart or identified using well-known techniques such as linkage topromoter-less “reporter” genes). The AAV rep-cap genes (with p5replaced), preferably linked to a positive selectable marker such as anantibiotic resistance gene, are then stably integrated into a suitablehost cell (such as the HeLa or A549 cells exemplified below). Cells thatare able to grow relatively well under selection conditions (e.g. in thepresence of the antibiotic) are then tested for their ability to expressthe rep and cap genes upon addition of a helper virus. As an initialtest for rep and/or cap expression, cells can be readily screened usingimmunofluorescence to detect Rep and/or Cap proteins. Confirmation ofpackaging capabilities and efficiencies can then be determined byfunctional tests for replication and packaging of incoming rAAV vectors.Using this methodology, a helper-virus-inducible promoter derived fromthe mouse metallothionein gene has been identified as a suitablereplacement for the p5 promoter, and used for producing high titers ofrAAV particles (as described in WO 96/17947, Targeted GeneticsCorporation).

Given the relative encapsidation size limits of various AAV genomes,insertion of a large heterologous polynucleotide into the genomenecessitates removal of a portion of the AAV sequence. Removal of one ormore AAV genes is in any case desirable, to reduce the likelihood ofgenerating replication-competent AAV (“RCA”). Accordingly, encoding orpromoter sequences for rep, cap, or both, are preferably removed, sincethe functions provided by these genes can be provided in trans.

The resultant vector is referred to as being “defective” in thesefunctions. In order to replicate and package the vector, the missingfunctions are complemented with a packaging gene, or a pluralitythereof, which together encode the necessary functions for the variousmissing rep and/or cap gene products. The packaging genes or genecassettes are preferably not flanked by AAV ITRs and preferably do notshare any substantial homology with the rAAV genome. Thus, in order tominimize homologous recombination during replication between the vectorsequence and separately provided packaging genes, it is desirable toavoid overlap of the two polynucleotide sequences. The level of homologyand corresponding frequency of recombination increase with increasinglength of the homologous sequences and with their level of sharedidentity. The level of homology that will pose a concern in a givensystem can be determined theoretically and confirmed experimentally, asis known in the art. Typically, however, recombination can besubstantially reduced or eliminated if the overlapping sequence is lessthan about a 25 nucleotide sequence if it is at least 80% identical overits entire length, or less than about a 50 nucleotide sequence if it isat least 70% identical over its entire length. Of course, even lowerlevels of homology are preferable since they will further reduce thelikelihood of recombination. It appears that, even without anyoverlapping homology, there is some residual frequency of generatingRCA. Even further reductions in the frequency of generating RCA (e.g. bynonhomologous recombination) can be obtained by “splitting” thereplication and encapsidation functions of AAV, as described by Allen etal. in U.S. patent application Ser. No. 08/769,728, filed 18 Dec. 1996,published internationally as WO98/27204 on 25 Jun. 1998 (TargetedGenetics Corporation)).

The rAAV vector construct, and the complementary packaging geneconstructs can be implemented in this invention in a number of differentforms. Viral particles, plasmids, and stably transformed host cells canall be used to introduce such constructs into the packaging cell, eithertransiently or stably.

In certain embodiments of this invention, the AAV vector andcomplementary packaging gene(s), if any, are provided in the form ofbacterial plasmids, AAV particles, or any combination thereof. In otherembodiments, either the AAV vector sequence, the packaging gene(s), orboth, are provided in the form of genetically altered (preferablyinheritably altered) eukaryotic cells. The development of host cellsinheritably altered to express the AAV vector sequence, AAV packaginggenes, or both, provides an established source of the material that isexpressed at a reliable level.

A variety of different genetically altered cells can thus be used in thecontext of this invention. By way of illustration, a mammalian host cellmay be used with at least one intact copy of a stably integrated rAAVvector. An AAV packaging plasmid comprising at least an AAV rep geneoperably linked to a promoter can be used to supply replicationfunctions (as described in a co-owned application by Flotte et al., nowU.S. Pat. No. 5,658,776). Alternatively, a stable mammalian cell linewith an AAV rep gene operably linked to a promoter can be used to supplyreplication functions (see, e.g., Trempe et al., (U.S. Ser. No.08/362,608, 9 Jan. 1995, WO95/13392, 18 May 1995); Burstein et al.,(U.S. Ser. No. 08/770,122, filed 18 Dec. 1996, WO98/23018, 25 Jun.1998); and Johnson et al., (U.S. Ser. No. 08/254,358, filed 6 Jun. 1994,issued as U.S. Pat. No. 5,656,785, 19 Aug. 1997)). The AAV cap gene,providing the encapsidation proteins as described above, can be providedtogether with an AAV rep gene or separately (see, e.g., theabove-referenced applications and patents as well as Allen et al., U.S.Ser. No. 08/769,728, filed 18 Dec. 1996, WO98/27204 on 25 Jun. 1998(Targeted Genetics Corporation)). Other combinations are possible andincluded within the scope of this invention.

Introduction of Genetic Material Into Cells

As is described in the art, and illustrated both herein and in thereferences cited above, genetic material can be introduced into cells(such as mammalian “producer” cells for the production of AAV) using anyof a variety of means to transform or transduce such cells. By way ofillustration, such techniques include for example transfection withbacterial plasmids, infection with viral vectors, electroporation,calcium phosphate precipitation, and introduction using any of a varietyof lipid-based compositions (a process often referred to as“lipofection”). Methods and compositions for performing these techniqueshave been described in the art and are widely available.

Selection of suitably altered cells may be conducted by any technique inthe art. For example, the polynucleotide sequences used to alter thecell may be introduced simultaneously with or operably linked to one ormore detectable or selectable markers as is known in the art. By way ofillustration, one can employ a drug resistance gene as a selectablemarker. Drug resistant cells can be picked and grown, and then testedfor expression of the desired sequence—i.e., a packaging gene product,or a product of the heterologous polynucleotide, as appropriate. Testingfor acquisition, localization and/or maintenance of an introducedpolynucleotide can be performed using DNA hybridization-based techniques(such as Southern blotting and other procedures as known in the art).Testing for expression can be readily performed by Northern analysis ofRNA extracted from the genetically altered cells, or by indirectimmunofluorescence for the corresponding gene product. Testing andconfirmation of packaging capabilities and efficiencies can be obtainedby introducing to the cell the remaining functional components of AAVand a helper virus, to test for production of AAV particles. Where acell is inheritably altered with a plurality of polynucleotideconstructs, it is generally more convenient (though not essential) tointroduce them to the cell separately, and validate each step seriatim.References describing such techniques include those cited herein.

Selection and Preparation of Helper Virus.

As discussed above, AAV is a parvovirus that is defective forself-replication, and must generally rely on a helper virus to supplycertain replicative functions. A number of such helper viruses have beenidentified, including adenoviruses, herpes viruses (including but notlimited to HSV1, cytomegalovirus and HHV-6), and pox viruses(particularly vaccinia). Any such virus may be used with this invention.

Frequently, the helper virus will be an adenovirus of a type andsubgroup that can infect the intended host cell. Human adenovirus ofsubgroup C, particularly serotypes 1, 2, 4, 6, and 7, are commonly used.Serotype 5 is generally preferred.

The features and growth patterns of adenovirus are known in the art. Thereader may refer, for example, to Horowitz, “Adenoviridae and theirreplication”, pp 771-816 in “Fundamental Virology”, Fields et al., eds.The packaged adenovirus genome is a linear DNA molecule, linked throughadenovirus ITRs at the left- and right-hand termini through a terminalprotein complex to form a circle. Control and encoding regions forearly, intermediate, and late components overlap within the genome.Early region genes are implicated in replication of the adenovirusgenome, and are grouped depending on their location into the E1, E2, E3,and E4 regions.

Although not essential, in principle it is desirable that the helpervirus strain be defective for replication in the subject ultimately toreceive the genetic therapy. Thus, any residual helper virus present inan rAAV preparation will be replication-incompetent. Adenoviruses fromwhich the E1A or both the E1A and the E3 region have been removed arenot infectious for most human cells. They can be replicated in apermissive cell line (e.g. the human 293 cell line) which is capable ofcomplementing the missing activity. Regions of adenovirus that appear tobe associated with helper function, as well as regions that do not, havebeen identified and described in the art (see, e.g., P. Colosi et al.,WO97/17458, and references cited therein).

Use of a Conditionally-Sensitive Helper Virus

As described herein, a “conditionally-sensitive” helper virus can alsobe employed to provide helper virus activity. Such a helper virus strainmust minimally have the property of being able to support AAVreplication in a host cell under at least one set of conditions where ititself does not undergo efficient genomic replication. Where helpervirus activity is supplied as intact virus particles, it is alsogenerally necessary that the virus be capable of replication in a hostcell under a second set of conditions. The first set of conditions willdiffer from the second set of conditions by a readily controllablefeature, such as the presence or absence of a required cofactor (such asa cation), the presence or absence of an inhibitory drug, or a shift inan environmental condition such as temperature. Most conveniently, thedifference between the two conditions is temperature, and such aconditionally-sensitive virus is thus referred to as atemperature-sensitive helper virus (tsHV).

For the purposes of this disclosure, a “temperature-sensitive” or “ts”helper virus is one which is capable of replicating its genetic materialin a eukaryotic cell at a certain temperature range (the “permissive”temperature range), typically about 15°-35° C. and preferably about20-32° C. However, at the “non-permissive” temperature, even when otherconditions are kept the same, the rate of replication of geneticmaterial is substantially lower, at least 10-fold lower, usually atleast about 100-fold lower; and preferably at least about 1000-foldlower. This temperature is typically about 35°-50° C., generally about42° C. In a typical example of such a ts helper virus, the virus iscapable of efficient replication at relatively low temperatures such astemperatures of about 20-32° C., but is incapable of efficientreplication at relatively high temperatures such as temperatures ofabout 37-42° C. It is understood that the virus-infected cell maynonetheless exhibit some metabolic processes attributable to the virusat the non-permissive temperature, including but not limited to helperfunction for AAV production.

A temperature-sensitive helper virus can be produced in bulk quantitiesby culturing infected cells at a permissive temperature. AAV vector canthen be produced by culturing cells comprising vector elements and thetemperature-sensitive helper virus at a non-permissive temperature. Thevector preparation will be substantially free of helper viruscomponents.

A large number of temperature-sensitive adenovirus variants have beendescribed in the art; see, e.g., the variants described by Ensinger etal. (J. Virol. 10:328, 1972); Williams et al. (J. Gen Virol. 11:95,1971); Ishibashi (Proc. Natl. Acad. Sci. USA 65:304, 1970); Lundholm etal. (Virology 45:827, 1971), and Shiroki et al., (Virology 61:474,1974); amongst others. Complementation analysis indicates that suchvariants fall into a plurality of different complementation groups(Ginsberg et al., Cold Spring Harbor Symp. Quant. Biol. 34:419, 1974).This suggests that a number of steps in the adenovirus replicative cyclemay be rendered temperature-sensitive.

Since helper function for AAV replication requires that only part of theadenovirus cycle be intact, testing for helper function of variousmutants at the non-permissive temperature provides a means for mappingthe helper function. For example, Ishibashi et al. (Virology 45:317,1971) reported that temperature-sensitive avian adenovirus variantssupport replication of AAV1 and AAV2. Ito et al. reported thattemperature-sensitive mutant ts13 of human adenovirus 7 (Ad7ts13) helpsAAV replication at the non-permissive temperature as efficiently as thewild strain. Drake et al. (Virology 60: 230, 1974) reportedcomplementation of AAV4 antigen synthesis by 3 groups oftemperature-sensitive mutants of herpes simplex virus type 1 (HSV1).Handa et al. (J. Gen. Viro. 29:239, 1975) reported helper activity forAAV1 virus production by human adenovirus mutants Ad5ts36, Ad5ts125,Ad5ts149, Ad12tsA275, Ad12ts.B221, and Ad12tsC295. Ostrove et al.(Virology 104:502, 1980) reported that temperature sensitive mutantsAd5ts125, Ad5ts135, Ad5ts157, Ad5ts116, and Ad5ts142, and the host rangemutants hr6 but not hr3 support AAV replication. Mayor et al. (J. GenVirol. 35:545, 1977) reported that Ad31ts13 but not Ad31ts94 supportedAAV1 production at the non-permissive temperature.

Straus et al. (Proc. Natl. Acad. Sci. USA 73:742, 1976) reported thatAd5ts125 supported AAV2 replication under conditions where theadenovirus did not itself replicate. They used this property to studyDNA intermediates formed during AAV replication. Myers et al. (J. Virol.35:65, 1980) performed a quantitative study on helper function, andshowed that Ad5ts149 supported the production of 20,000 infectious AAVparticles per cell at the non-permissive temperature, whereas Ad5ts107produced only ˜100 particles per cell. Since Ad5ts107 has a mutation inthe 72 kDa DNA binding protein encoding region, they concluded that thisprotein played a role in the AAV RNA expression. More recently, Carteret al. (Virology 191:473, 1992) proposed that a fully functional 72 kDaprotein is required for quantitative post-transcriptional expression ofthe AAV rep and cap genes.

As outlined in the background section, the existence oftemperature-sensitive adenovirus has been known for quite some time.However, there has been no effective teaching or suggestion regardingthe actual use of conditional helper viruses in the generation ofrecombinant AAV vectors, such as those that might be used for genetherapy.

Part of the explanation may be the difficulty in obtaining workabletiters of AAV when using recombinant vectors. Among other things, AAVRep proteins apparently down-regulate their own expression through thep5 promoter (Tratschin et al., Mol. Cell Biol. 6:2884, 1986). Inaddition, it has been observed that the expression of the rep gene inpackaging cell lines such as those that might be used for the productionof recombinant AAV vector, tends to inhibit the growth and/or metabolismof the cell (see, e.g., Targeted Genetics Corporation, WO96/17947, byAllen et al.).

The differences between the generation of wild-type AAV and recombinantAAV vectors tend to be quite dramatic when considered in terms ofproduction. In particular, it has been observed that production ofrecombinant AAV vectors tends to be substantially lower that productionof wild-type AAV particles, and that the presence or generation of evensmall amounts of contaminating wild-type AAV tends to result in apreferential production of wild-type virus that can eventually outnumberthe recombinant AAV vectors.

These phenomena are further illustrated by the results described inExamples 1 and 2 of this disclosure and in FIG. 1. The adenovirustemperature-sensitive mutant ts149 is reported elsewhere to support AAVparticle replication (Myers et al., J. Virol. 35:65, 1980). However,Example 2 shows that when this mutant is used to support the productionof an AAV vector with a heterologous promoter under standard conditions,the level of production is several orders of magnitude lower than issupported by wild-type adenovirus.

This disclosure shows that temperature-sensitive helper virus can indeedbe used to prepare recombinant AAV vectors at workable titers,overcoming the apparent production obstacles. The descriptions thatfollow illustrate how to select a temperature-sensitive helper virus andoptimize conditions to provide sufficient AAV for the purposes of genetherapy.

In particular, it is shown that extending the replication period for AAVwhen using tsAd as helper dramatically increases the amount of AAVvector that is produced (Example 3). This is counter-intuitive, becauseextending the replication period when using wild-type Ad in the same waydecreases the amount of AAV vector by at least an order of magnitude. Apractitioner of skill in the art seeking to optimize conditions for AAVproduction would logically go to shorter culture times and higherconcentrations of helper virus; both of which are shown herein to beineffective.

This invention further provides improved culture and separation methodsfor preparing quantitative amounts of temperature-sensitive adenovirus.While not strictly required for the practice of certain embodiments ofthis invention, preparations of temperature-sensitive adenovirusobtained by these methods are particularly suited for production of AAV,inter alia, for the purposes of gene therapy.

Condition-sensitive variants of the selected strain of helper virus maybe generated by an appropriate mutagenization and selection strategy.For example, virus may be mutagenized with nitrosoguanidine, nitrousacid, hydroxylamine, or 5-bromo-2-deoxyuridine. Candidates are selectedthat can multiply in a suitable eukaryotic cell under the desiredpermissive conditions, but not under the desired non-permissiveconditions. As an illustration, adenovirus temperature-sensitive mutantscan be obtained that multiply, e.g., at 32° C., but not at 39.5° C.Plaquing efficiency ratios at 39.5° C. versus 32° C. are preferably lessthan 10⁻⁴ and more preferably less than 10⁻⁵. Further illustration ofsuitable selection processes for temperature-sensitive adenovirus can befound, for example, in Ensinger et al., J. Virol. 10:328, 1972; andWilliams et al., J. Gen Virol. 11:95, 1971. Description of adenovirusvariants which are not temperature-sensitive, but host-range sensitive,can be found in Harrison et al., Virology 77:319, 1977.Temperature-sensitive mutants effective for use in this invention can beprepared, for example, from alternative helper viruses like herpessimplex 1 (HSV1), or herpes simplex 2 (HSV2). See, e.g., Schaffer etal., Virology 52:57, 1973 for HSV1; Esparza et al., Virology 57:554,1974 for HSV2. As indicated in the background section, a large number ofcondition-sensitive helper viruses have been described, and can beobtained from the scientists who developed or described them or from apublic depository.

Not all condition-sensitive variants of the aforelisted viruses willwork with the present invention. In particular, the strain must berendered condition-sensitive at a stage in its replicative cycle suchthat the function that is blocked under non-permissive conditions is notone that is required for high-efficiency replication of AAV. The choiceof which helper virus strain to use can be made by reference to both theknown biology of the helper virus and the replicative requirements ofAAV.

An exemplary helper virus for use with this invention is thetemperature-sensitive adenovirus ts149 of the Ad5 serotype (Ad5ts149).As shown in the example section, under optimized conditions, this straincan be used to produce rAAV at levels that match or exceed thosesupported by wild-type Ad5. The ts149 has a single transition of C-G toA-T at position 7563 (Roovers et al., Virus Genes 4:53, 1990). Thisresults in a change of amino acid leucine at residue 411 of the DNApolymerase to phenylalanine. The DNA polymerase is contained within theE2 transcription unit of adenovirus. However, other ts mutants mappingto this region are less suitable. In particular, the E2 transcriptionunit also comprises the encoding region for the 72 kDa DNA bindingprotein (DBP). A strain that produces no detectable DBP (Add/802)supports AAV replication, but at a level that is reduced by an order ofmagnitude (Carter et al., Virology 191:473, 1992). Adts125, which alsocomprises a mutation mapping to the DBP encoding region, support AAVreplication (Straus et al., J. Virol. 17:140, 1976), although the levelsare generally much lower than with wild-type Ad5 (Myers et al., J.Virol. 35:65, 1980). Accordingly, suitable temperature-sensitiveadenovirus vectors for use in this invention include those for which thesensitivity maps to the E2A region of the genome, preferably to the DNApolymerase encoding region.

The artisan can readily determine which viral strains are suitable foruse as helper virus by conducting an rAAV replication assay using apanel of candidate helper virus strains in a candidate cell underconditions that are non-permissive for self-replication of the helper.For temperature-sensitive variants, screening is done at thenon-permissive temperature according to the known properties of thestrain. Non-permissive temperatures are generally higher than permissivetemperatures, typically about 35°-50° C., preferably 38°-45° C., morepreferably about 39.5° C. Variants supporting AAV replication at a levelthat is within one order of magnitude of that supported by thecorresponding wild-type virus is preferred. In conducting the screening,the artisan should incorporate the other teachings of this disclosure.In particular, screening by culturing for times that give peak AAVreplication with wild-type virus is insufficient. A kinetic matrixshould be set up in which the candidate helper viruses are used forlonger periods, and then compared with the wild-type virus at peakharvest time. A more detailed illustration of this analysis is providedin Example 3 of this disclosure.

Once a suitable helper virus strain has been selected, it may beimplemented in this invention in a number of different forms. Viralparticles, viral plasmids, and stably transformed host cells can all beused.

In one embodiment, the genome of the helper virus (or minimally, theregions of the helper virus genome encoding helper function) isintroduced into the host cell to be used for replication of the rAAVvector in the form of a DNA plasmid, or a plurality of plasmids thatprovide complementary functions. Procedures for experimentalmanipulation of adenovirus are known in the art. The reader is referredto Graham et al., “Manipulation of adenovirus vectors”. In: Murray E J,ed Methods in molecular biology: Gene transfer and expression protocols,vol7. Clifton, N.J.: The Human Press, 1991:109-128, which providesdetailed protocols for propagation, titration, and purification ofadenovirus, cotransfection and in vivo recombination. Adenovirusplasmids are available commercially from Microbix Biosystems Inc.,Toronto, Canada.

In another embodiment, the host cell is stably-transfected withadenovirus genes, or genetically altered to provide the requisitefunctions for rAAV replication. Alternatively, the host cell may begenetically altered with only a portion of the adenovirus genome, and issubsequently infected or transfected with an adenovirus particle orplasmid. Patent applications WO 95/27071 and WO 95/34671 describe hostcells inheritably altered to provide adenovirus function, whichcomplements the replicative property of various defective adenovirusconstructs.

In yet another embodiment, the host cell used for AAV replication isinfected with a helper virus which is capable of self-replication, butnot under non-permissive conditions. Any preparation of the requisitestrain providing a sufficient MOI may be used. In keeping with GMP andother regulatory requirements, and to facilitate scale-up for commercialpurposes, preparations of helper virus preferably comprise a highdensity of infectious particles and are substantially free of cellulardebris and other contaminants. Desirable properties include thefollowing:

-   -   A density of at least 10⁶, preferably at least about 10⁸, more        preferably at least about 10¹⁰ IU/ml, as determined in a TCID₅₀        assay.    -   A ratio of adenovirus DNA to total protein or adenovirus hexon        that indicates that at least 10%, preferably at least about 50%,        more preferably at least about 80% of the viral particles        contain adenovirus DNA.    -   Less than 20%, preferably less than about 10%, more preferably        less than about 1% contamination by non-adenovirus material at        the protein or DNA level, as detected by SDS gels stained for        protein, or agarose gels of restriction nuclease digests stained        with ethidium bromide.    -   A total of at least 10⁹, preferably at least about 10¹¹, more        preferably at least about 10¹³ IU per production batch.

Helper virus may be prepared in any cell that is permissive for viralreplication. For adenovirus, preferred cells include 293 cells and HeLacells. Traditionally, when these cells have been used for replication ofadenovirus, they have been used in plate cultures. However, as shown inExample 4, these methods generally support replication oftemperature-sensitive adenovirus at levels that are one or two logslower than for wild-type adenovirus.

Accordingly, it is preferable to employ culture techniques that permitan increase in seeding density. 293 cells and HeLa cell variants areavailable that have been adapted to suspension culture. HeLa ispreferable for reasons of cell growth, viability, and morphology insuspension. As shown in Example 5, these cells can be grown atsufficient density (2×10⁶ per ml) to make up for the lower replicationrate of the temperature-sensitive adenovirus strain. Once established,cells are infected with the virus and cultured at the permissivetemperature for a sufficient period; generally 3-7 days and typicallyabout 5 days.

Tangential flow filtration is a technique used in the art for processinglarge volumes of mammalian cells for the purpose of perfusing,concentrating, and harvesting them. See, e.g., Dorin et al., Biotechnol.Prog. 6:494, 1990; Maiorella et al., Biotechnol. Bioeng. 37:121, 1991.It is recommended that this technique be used with suspension culturesfor the preparation of helper virus for use in this invention. Example 5demonstrates that HeLa S3 cells withstand shear forces of 750-1500 sec³¹¹, permitting concentration of the cells and diafiltration of spentmedia.

Virus is harvested from the culture either from the spent media or bymicrofluidization of the cells. The level of helper virus produced inthe culture is typically at least 10⁷ IU/ml, and preferably at leastabout 3×10⁷ IU/ml.

Helper virus prepared according to the foregoing description may be useddirectly for infecting host cells used for rAAV replication. Moreusually, the virus is isolated and concentrated before use. Currentmethods for purifying and concentrating helper virus typically involveisopynic CsCl gradients. This method is time and labor intensive,requires numerous open processing steps, and is difficult to scale up.Instead, purification by chromatography is recommended. The reader isreferred generally to Prior et al., Pharmaceut. Technol. 19:30, 1995;and Huyghe et al., Human Gene Therapy 6:1403, 1995. Particularlypreferred for isolation of temperature-sensitive strains of adenovirusis anion exchange chromatography, especially on a resin ofpolyethyleneimine using a continuous NaCl gradient at pH 7.4. A detailedillustration of the polyethyleneimine separation method is provided inExample 6.

Providing a Host Cell (Producer Cell) Comprising Helper Virus Functionand AAV

In the methods of the invention, producer cells comprising componentsnecessary for viral replication and encapsidation are cultured (in someembodiments, under conditions that promote viral release). Severalcriteria influence selection of cells for use in producing rAAVparticles as described herein. As an initial matter, the cell must bepermissive for replication and packaging of the rAAV vector when usingthe selected helper virus. However, since most mammalian cells can beproductively infected by AAV, and many can also be infected by helperviruses such as adenovirus, it is clear that a large variety ofmammalian cells and cell lines effectively satisfy these criteria. Amongthese, the more preferred cells and cell lines are those that can beeasily grown in culture so as to facilitate large-scale production ofrecombinant AAV vector preparations. Again, however, many such cellseffectively satisfy this criterion. Where large-scale production isdesired, the choice of production method will also influence theselection of the host cell. For example, as described in more detailbelow and in the art, some production techniques and culture vessels orchambers are designed for growth of adherent or attached cells, whereasothers are designed for growth of cells in suspension. In the lattercase, the host cell would thus preferably be adapted or adaptable togrowth in suspension. However, even in the case of cells and cell linesthat are regarded as adherent or anchorage-dependent, it is possible (asdescribed below) to derive suspension-adapted variants of ananchorage-dependent parental line by serially selecting for cellscapable of growth in suspension. Developing and obtaining asuspension-adapted cell line which is also capable of producing highyields of virus provides a significant advantage.

Where a temperature-sensitive helper virus is used, the cell must beable to effectively replicate the rAAV vector under conditions that arenon-permissive for replication of the helper virus. By way ofillustration, when adenovirus ts149 is used as a ts helper virus (asdescribed and illustrated below), the cell must be capable of supportingrAAV replication and packaging at temperatures well above 32° C.,preferably about 39.5° C. Human 293 cells are an example of a cell linefulfilling these criteria but numerous other cells and cell lines arecapable of replicating rAAV at this relatively elevated temperature.

Ultimately, the helper virus, the rAAV vector sequence, and all AAVsequences needed for replication and packaging must be present in thesame cell. Where one or more AAV packaging genes are provided separatelyfrom the vector, a host cell is provided that comprises: (i) one or moreAAV packaging genes, wherein each said AAV packaging gene encodes an AAVreplication or encapsidation protein; (ii) a heterologous polynucleotideintroduced into said host cell using an rAAV vector or pro-vector,wherein said rAAV vector or pro-vector comprises said heterologouspolynucleotide flanked by at least one AAV ITR and is deficient in saidAAV packaging gene(s); and (iii) a helper virus or sequences encodingthe requisite helper virus functions. It should be noted, however, thatone or more of these elements may be combined on a single replicon. Byway of illustration, a helper virus can also comprise an rAAV pro-vectoror an AAV packaging gene.

The helper virus is preferably introduced into the cell culture at alevel sufficient to infect most of the cells in culture, but canotherwise be kept to a minimum in order to limit the amount of helpervirus present in the resulting preparation. A multiplicity of infectionor “MOI” of 1-100 may be used, but an MOI of 5-10 is typically adequate.

Similarly, if the AAV vector and/or packaging genes are transientlyintroduced into the packaging cell (as opposed to being stablyintroduced), they are preferably introduced at a level sufficient togenetically alter most of the cells in culture. Amounts generallyrequired are of the order of 10 μg per 10⁶ cells, if supplied as abacterial plasmid; or 10⁸ particles per 10⁵ cells, if supplied as an AAVparticle. Determination of an optimal amount is an exercise of routinetitration that is within the ordinary skill of the artisan.

These elements can be introduced into the cell, either simultaneously,or sequentially in any order. Where the cell is inheritably altered byany of the elements, the cell can be selected and allowed to proliferatebefore introducing the next element.

In one preferred embodiment, the helper virus is introduced last intothe cell to rescue and package a resident rAAV vector. The cell willgenerally already be supplemented to the extent necessary with AAVpackaging genes. Preferably, either the rAAV vector or the packaginggenes, and more preferably both are stably integrated into the cell. Itis readily appreciated that other combinations are possible. Suchcombinations are included within the scope of the invention.

Once the host cell is provided with the requisite elements, the cell iscultured under conditions that are permissive for the replication AAV,to allow replication and packaging of the rAAV vector. Culture time ispreferably adjusted to correspond to peak production levels, and istypically 3-6 days. Preferably, at least 100 viral particles areproduced per cell; more preferably at least about 1000 per cell, stillmore preferably at least about 10,000 per cell. Preferably, at least0.5×10⁶, more preferably at least about 1×10⁶, even more preferably atleast about 2×10⁶ RU/ml AAV vectors are produced per 2×10⁵ cells duringthe culture period. Optionally, large-scale production methods such assuspension culture and tangential flow filtration may be used. AAVparticles are then collected, and isolated from the cells used toprepare them.

Preparations of rAAV particles of the present invention preferablycomprise a high density of infectious AAV particles and aresubstantially free of helper virus, helper virus proteins and cellulardebris and other contaminants. Desirable properties include thefollowing:

-   -   A concentration of at least 10⁷, preferably at least about 10⁸,        more preferably at least about 10⁹ RU/ml, as determined in a        replication assay or quantitative hybridization comparison with        a known standard.    -   No more than 10³, preferably no more than about 10², more        preferably no more than about 10¹ infectious particles of helper        virus per 10⁸ RU of rAAV particles.    -   Less than 5%, preferably less than about 1%, more preferably        less than about 0.01%, even more preferably less than about        0.001% contamination by helper virus on a protein basis (wt/wt),        detected either by densitometric analysis of SDS gels, or by        immunoassay for helper virus specific protein (such as hexon or        penton-fiber of adenovirus).    -   Less than 5%, preferably less than about 1%, more preferably        less than about 0.01%, even more preferably less than about        0.001% contamination by helper virus or cellular protein        (wt/wt), detected either by densitometric analysis of SDS gels,        or by immunoassay for helper virus or cellular specific        proteins.    -   Preferably, the preparation is also substantially free of other        potential cellular components such as cellular lipids,        carbohydrates and/or nucleic acids.

The methods outlined in this disclosure are suitable for preparing smallexperimental batches, or preparative batches of 10-100 liters or more.For large scale batch preparations, the following property is alsodesirable:

-   -   A total of at least 10¹⁰, preferably 10¹², and more preferably        10¹⁴ RU of AAV vector particles in the preparation.

Optionally, rAAV vectors may be further processed to enrich for rAAVparticles, deplete helper virus particles, or otherwise render themsuitable for administration to a subject. Purification techniques mayinclude isopyric gradient centrifugation, and chromatographictechniques. Reduction of infectious helper virus activity may includeinactivation by heat treatment or by pH treatment as is known in theart. Other processes may include concentration, filtration,diafiltration, or mixing with a suitable buffer or pharmaceuticalexcipient. Preparations may be divided into unit dose and multi dosealiquots for distribution, which will retain the essentialcharacteristics of the batch, such as the homogeneity of antigenic andgenetic content, and the relative proportion of contaminating helpervirus.

Exemplary techniques for generating preparations of helper virus and AAVexhibiting various desirable properties as described above are providedin the following sections and in the subsequent examples.

Various methods for the determination of the infectious titer of a viralpreparation are known in the art. However, a preferred method for titerdetermination is a high-throughput titering assay as provided herein. Inan exemplary high-throughput titering assay, an array of culture wellseach comprising an aliquot of mammalian cells and an aliquot of viruspreparation (as well as control wells comprising e.g., cells alone,virus alone and null) is established. The array of culture wells may,for example, be in the form of a microtiter vessel. Typically, aliquots(e.g., serially diluted aliquots) of the virus preparation to be titeredare added to the cells, and then the cells and virus are incubated underconditions that allow for infection and replication of the virus(typically growth conditions suitable for the mammalian host cell).Following replication of the virus, viral nucleic acid is generallyreleased by lysis of the mammalian cells (using conditions or agentsthat promote lysis as necessary). In preferred embodiments, nucleic acid(including viral nucleic acid) in the multiplicity of lysates istransferred and fixed to a membrane under conditions that bind nucleicacid (washing as appropriate to remove proteins and other contaminants).The membrane preferably is a replicate or mirror image of the culturearray in which the individual wells of the original array aresubsequently represented by “pools” of nucleic acid (from the lysate ofeach culture well) that are bound at corresponding positions on themembrane. Hybridizing the membrane with a labeled virus-specific (orviral-insert-specific) probe can then be used to identify and quantifythe relative amount of viral-specific nucleic acid in each of the pointson the array, and by correspondence, in each of the original culturewells. Conditions and materials for nucleic acid transfer, binding,washing and hybridizing can be adapted from routine molecular biologicaltechniques such as “dot blot” hybridization (as described in the art,see, e.g. the molecular biological techniques in Sambrook et al., supra,and Ausubel et al., supra). Illustrative applications of thesetechniques are presented below.

These methods thus provide a high-throughput infectivity assay which canbe used in the determination of the infectious titer of a viruspreparation. As shown in Example 4, virus titers determined by thisrapid and quantitative method closely correspond to the titersdetermined by more classical techniques. In addition, however, thishigh-throughput method allows for the concurrent processing and analysisof many viral replication reactions and thus has many others uses,including for example the screening of cell lines permissive ornon-permissive for viral replication and infectivity, as well as thescreening of agents that affect viral infection and/or replication, asdiscussed further below.

Preferred Helper Virus Production and Purification Techniques for Use inthe Present Invention

In various preferred aspects of the present invention, production andpurification methods are employed for the generation of helper virussuitable for use in the production of rAAV vectors as described herein.A commonly used helper virus for the production of AAV is adenovirus,typically Ad5, although other helper viruses can also be employed asdiscussed herein and in the art.

For purposes of illustration, it is convenient to divide the discussionof virus production and purification into “upstream” and “downstream”phases. The “upstream” process generally refers to the production of thevirus in suitable host cells and release or removal of the virus fromthe cells to produce a “crude” virus preparation such as a lysate.“Downstream” processing can be employed to purify the crude viruspreparation (e.g. to isolate it away from cellular proteins and/or othercontaminants).

A variety of techniques are known for the production and processing ofhelper viruses, including adenovirus (e.g., CsCl centrifugation, as wellas other techniques such as those described in WO 96/27677). Helpervirus produced using such techniques can then be employed in theproduction of rAAV vectors as described herein.

The following sections describe, for purposes of illustration,techniques that can be employed for the production of adenovirusalthough other techniques are known in the art and can be employedherein.

(i) Helper Virus Upstream

Helper virus, such as Ad5, can be readily produced by infectingmammalian cells (e.g. human cells). In illustrative examples describedbelow, cells are grown in media and culture vessels suitable for growthof the host cell, concentrated prior to infection, and then infectedwith helper virus (e.g. at an MOI of 1-5) with gentle stirring.Following infection, cells can be resuspended in fresh medium andincubated for an additional period of time (typically about 2 days) inorder to allow for replication and packaging of the helper virus.Following incubation, cells can be harvested and lysed to release thehelper virus. Following lysis, the cell lysate is preferably treatedwith a nuclease to degrade free nucleic acid (e.g. cellular nucleicacid) without degrading nucleic acid that is encapsidated in viralparticles. The lysate can be clarified (e.g. by filtration and/orcentifugation), and can also be subjected to further purificationtechniques in order to purify and concentrate the helper virus in thepreparation, as described and illustrated below. In some embodiments,the lysate is subjected to filtration (such as depth filtration) toclarify the lysate, followed by heat killing, followed by filtration(such as filtration using a 0.5 μm filter) to further clarify thelysate, followed by cation exchange chromatography (using, for example,an HS resin), followed by nuclease digestion, followed by anion exchangechromatography (using, for example, a PI resin), followed by heparinsulfate chromatography, followed by gel filtration.

As an illustrative example of such a process, cells can be grown inmedia at a density of about 1×10⁶ cell/ml in a vessel such as a spinnerflask. After incubation, cells can then be concentrated to about 10⁷cells/ml, and infected with Ad5 at 1-2 infectious units/cell with gentlestirring. Cells can then be resuspended in medium at about 10⁶ cells/ml,and allowed to produce virus over an incubation period of about 2 days.Cells can then be harvested, resuspended in medium or buffer (e.g., atabout 5×10⁶ cells/ml), and then disrupted, e.g. by mechanical lysis suchas by passaging through a microfluidizer at 8000 psi or equivalenttechnique (e.g. freeze-thaw or sonication). The lysate can be treatedwith a nuclease (e.g., Benzonase) for one hour at 37° C. The lysate canbe clarified through a filter, such as a 1.0μ filter, or bycentrifugation. Analogous techniques and modifications thereof arefurther described below.

(ii) Helper Virus Downstream

Preferred techniques for the downstream processing of helper virus, suchas adenovirus, employ ion-exchange chromatographic procedures for thepurification of the helper virus.

By way of illustration, the adenovirus filtrate as described above canbe loaded on an anion-exchange resin, such as an N-charged amino orimino resin (e.g. POROS 50 PI, or any DEAE, TMAE, tertiary or quaternaryamine, or PEI-based resin) in a chromatography column equilibrated withbuffer (such as TMEG, also referred to herein as Chromatography BufferA: 50 mM Tris (pH 8.0), 5 mM MgCl₂, 1 mM EDTA, 5% glycerol).

The column can then be washed with multiple column volumes of TMEG (e.g.5-6 volumes), followed by multiple volumes of a saline wash (e.g. 5-6volumes of TMEG with 800 mM NaCl (Chromatography Buffer “B”: 60% TMEGand 40% TMEG with 2M NaCl). The Adenovirus can be eluted with TMEG with1300 mM NaCl. (35% Chromatography Buffer A, 65% Chromatography BufferB).

The peak of adenovirus can be identified in the fractions by aninfectivity assay or by a nucleic acid hybridization or immunoassay, ashave been described in the art. The peak can be sterile filtered througha 0.2μ sterile filter. Optionally, the peak can be concentrated bytangential-flow filtration, for example in a Filtron Ultrasette orMillipore Pellicon unit. The peak or concentrate may be diafiltered inthis system into a suitable buffer, such as PBS+5% Sucrose.Alternatively, the adenovirus can be left in elution buffer. The finaladenovirus product can be sterile filtered through a 0.2μ filter andstored for use. As described and illustrated herein, atemperature-sensitive helper virus (such as a temperature-sensitiveadenovirus) can also be employed.

Examples describing the preparation and use of such helper viruses areprovided below for purposes of further illustration.

Preferred AAV Production and Purification Techniques For Use in thePresent Invention

As with helper virus, it is convenient for purposes of illustration todivide the discussion of AAV production and purification into “upstream”and “downstream” process phases; with the “upstream” process generallyreferring to the production of AAV in suitable host cells and release orremoval of the virus from the cells to produce a “crude” AAVpreparation. “Downstream” processing can be employed to purify the AAVpreparation (e.g. to isolate AAV away from cellular proteins and/orother contaminants).

In preferred aspects of the present invention, upstream and downstreamprocessing of AAV are conducted in a manner designed to substantiallyreduce and/or eliminate contaminating cellular proteins, as well as anycontaminating helper virus (e.g. Ad) or helper virus proteins, any ofwhich might contribute to elicitation of an immune response if presentat substantial levels in the final rAAV vector preparation to be usedfor gene transfer.

The following sections describe, for purposes of illustration,techniques that can be employed for the production of AAV.

(i) AAV Upstream Processing

AAV vector can be produced from a mammalian cell line that contains thenecessary AAV packaging genes (e.g. an AAV rep and cap gene); arecombinant AAV (rAAV) pro-vector that comprises a heterologous non-AAVpolynucleotide flanked by at least one AAV inverted terminal repeat(ITR); and a helper virus for AAV (e.g. an adenovirus). These componentscan be introduced into the cell in a variety of configurations, asdescribed above and illustrated below. Since AAV can be replicated andpackaged in any of a variety of mammalian cells, there are a largenumber of cell lines that can be modified and employed for theproduction of AAV.

By way of illustration, AAV vector can be produced from a cell line,such as “C12” (as described by K. R. Clark et al., Gene Therapy, 3:1124-1132, 1996) or the “C137.5” line (described in a commonly-ownedcopending application by Targeted Genetics Corporation, J. Allen et al.,WO 96/17947), that has been engineered to contain a rep and/or a capconstruct, as well as a vector construct. Optionally, a cell line suchas C12 or c137 that contains a rep and/or a cap construct can betransfected with a plasmid that contains a vector construct, such asptgAAV-CF. Or a cell can be transfected with a plasmid that contains repand cap, such as pRS5, as well as a plasmid that contains a vectorconstruct. The cell can be infected with Adenovirus, or transfected withDNA that contains adenovirus genes.

A variety of such AAV “producer” cells can be generated, as described inthe references cited herein and in the art.

The AAV producer cells can be grown under conditions (including media,temperature and the like) that are generally suitable for growth of themammalian cells, which are generally also permissive for the replicationof AAV. For example, DMEM/F12 suspension medium is preferred for growthof the cells and DMEM medium alone is preferred for AAV vectorproduction. As is known in the art, some cell types and cell lines tendto be attachment-dependent, whereas others are capable of growth insuspension; and many attachment-dependent cells can also be “adapted” togrowth in suspension by cycling of the cells under suspension conditionsas a means of enriching for and ultimately selecting for variants thatare capable of suspension growth. Accordingly, the invention providesmethods of generating a population of rAAV particles comprising the stepof incubating a producer cell in a cell culture medium, wherein saidproducer cell is cultured under suspension conditions, whereby rAAVparticles are produced. For these embodiments, the producer cell isother than a KB cell. Carter et al. (1979) Virology 92:449462; Tratschinet al. (1985) Mol. Cell. Biol. 5(11):3251-3260. Levels of productionusing suspension cultures of the invention has been very high (e.g.,greater than about 10², 10 ³ or even 10⁴ particles per cell on averagein a given cell population). Examples of cells which may be grown insuspension are described herein. In some embodiments, cells produceabout 10² to about 10⁴ virus particles per cell; in other embodiments,about 10² to about 10³ virus particles per cell; in other embodiments,about 10³ to about 10⁵ virus particles per cell; in other embodiments,about 10⁴ to about 10⁶ virus particles per cell. Growth of cells for AAVproduction can be conducted in any of a variety of vessels, depending inpart on whether the selected producer cell line is relatively attachmentdependent or is suspension adapted. Such vessels for the growth ofattachment-dependent cells include, for example, tissue culture flasks,roller bottles, microcarriers and bioreactors (such as hollow-fiber,packed-bed or fluidized-bed bioreactors). Vessels for suspension-adaptedmammalian cell lines include, for example, spinner flasks, tank reactorsand air lift fermentors.

AAV replication proceeds for a period of time as well as to a point inthe growth cycle where viral production is optimal, preferable mid- tolate-logarithmic growth (typically one to three days), after which timethe cells can be harvested and lysed to release progeny virus. Forexample, cells can be resuspended in growth media to about 1-10×10⁶cells/ml, and allowed to produce for 48 hours. Cells can then beharvested (e.g. by centrifugation), and resuspended in buffer (e.g.,TMEG (or “Chromatography Buffer A”): 50 mM Tris, pH 8.0, 5 mM MgCl₂, 1mM EDTA, 5% Glycerol) at about 1-10×10⁶ cells/ml.

AAV can replicate to high copy number (e.g. 10⁵-10⁶ genomes/cell) intransduced cells if the necessary AAV Rep proteins and helper virusfunctions are provided relatively simultaneously. If Cap proteins arealso provided, AAV particles are assembled in the nucleus of theinfected cells where they tend to be assembled in crystalline arrays.The first step in product recovery is therefore generally cellulardisruption, except in those embodiments which involve culturing cellsunder conditions which promote release of virus. It is understood,however, that embodiments which involve release of virus may alsoinclude a cell lysis step. Although freeze-thawing and/or sonication canbe used to disrupt the cells (as with adenovirus), such techniques arenot very suitable to large-scale preparation. Mechanical lysis, usingtechniques such as microfluidization are thus preferable in thoseregards. Detergents and other chemical agents can also be employed tomediate or facilitate lysis. Treatment of lysates with nucleases (suchas Benzonase) has been found to be helpful for reducing viscosity andimproving filterability. Clarification, e.g. by microfiltration toseparate vector from at least some portion of the cellular debris, isalso helpful for promoting recovery and purification.

By way of illustration, cells can be mechanically lysed after theincubation period by sequential passaging through a microfluidizer(typically at about 8000 psi, using two passages). Othercommonly-employed techniques include freeze-thaw cycling and sonication,as is known in the art. The lysate can also be treated with a nucleaseto degrade nucleic acid (such as cellular or viral nucleic acid) that isnot effectively “protected” by virtue of being packaged into a viralparticle. We typically employ Benzonase digestion for about one hour at37° C. The lysate can also be clarified. Methods for clarificationinclude passage through a filter, such as a 1.0μ filter, andcentrifugation.

Tangential flow filtration (TFF) can be beneficially employed forprocessing and harvesting large volumes of cells. TFF can be used toperfuse, concentrate and harvest animal cells. For example, TFF can beused to process cells under laminar flow conditions at average wallshear rates of up to 3000 per second (see, e.g., Maiorella, B., et al.,Biotechnology and Bioengineering, 37: 121-126, 1991). Large-scaleconcentration of viruses using TFF ultrafiltration has been described byR. Paul et al. Human Gene Therapy, 4:609-615, 1993.

If lysis if not required or indicated, cells may be removed from culturemedium to provide culture supernatant containing virus particles usingmethods standard in the art, such as centrifugation and/or filtration.

Illustrative production runs employing such techniques are describedbelow.

(ii) AAV Downstream Processing

As described above, it would be particularly advantageous to obtainpreparations of AAV that are substantially free of helper virusparticles (such as Ad particles). In addition, AAV vector preparationswill preferably also be substantially free of helper virus and cellularproteins (which can also be immunogenic). However, there is a furtherset of constraints that influence the suitability of techniques for AAVproduction. Namely, in order to be particularly useful for theproduction of AAV for gene therapy, it is most desirable for thetechniques to be “scalable”, i.e. applicable in conjunction withlarge-scale manufacturing devices and procedures. This latter set ofconstraints effectively reduces or eliminates the utility of availablestandard techniques such as cesium chloride separation (which is notwell-suited to large-scale preparation procedures).

We have discovered a combination of procedures that are both scalableand remarkably effective for the generation of AAV preparations that aresubstantially free of helper virus particles, as well as helper virusand cellular proteins and other such contaminants. Our preferredcombination of procedures employs ion exchange chromatographicprocedures which contrast with various procedures mentioned in the artfor the potential purification of, e.g., AAV or Ad. In particular, suchprocedures as described in the art typically employ a single type ofionic separation, sometimes in combination with other sorts ofchromatographic procedures (see, e.g., K. Tamayose et al., Human GeneTherapy 7: 507-513 (1996), and WO96/27677, Sep. 12, 1996). However, inthe case of AAV production, we have found that a combination ofsequential opposing ion exchange chromatography is particularlyeffective for the generation of AAV preparations that are substantiallyfree of helper virus particles and proteins, as well as cellularproteins. These opposing ion exchange chromatography steps may be in anyorder, and may include additional opposing ion exchange chromatographystep(s). For example, in some embodiments, a lysate or culturesupernatant is subjected to cation exchange chromatography followed byanion exchange chromatography followed by cation exchangechromatography. Preferably, heparin sulfate is used in at least one(preferably the last) cation exchange chromatography.

In view of these discoveries, it appears that AAV is not only “adapted”to both anion exchange and cation exchange chromatography, but that sucha combination of both opposing ionic exchanges is particularly effectivefor eliminating all of the various particle and protein contaminantsthat typically occur in the generation or AAV vector preparations. Anyof a variety of cation and anion exchange resins can be employed inconjunction with these procedures, the fundamental properties of whichare the availability of negatively- and positively-charged groups,respectively, to which AAV can bind at least to some degree (mostpreferably to a degree that differs substantially from the relativebinding affinity of one or more of the contaminants referred to above,i.e. Ad particles and proteins, as well as mammalian cellular proteins).Without wishing to be bound by theory, it is believed that the anionicexchange step is particularly effective for separating AAV fromAdenovirus; whereas both steps (but especially the cationic exchangestep) are believed to be particularly effective for separating AAV fromcellular proteins. We have also employed anion exchange followed bytangential flow filtration, as described and illustrated below. Asfurther described below, we have found AAV preparations can be highlyconcentrated by chromatography on heparin sulfate.

By way of illustration, a clarified AAV lysate as described above can beloaded on an positively charged anion-exchange column, such as anN-charged amino or imino resin (e.g. POROS 50 PI, or any DEAE, IMAE,tertiary or quaternary amine, or PEI-based resin) or a negativelycharged cation-exchange column (such as HS, SP, CM or any sulfo-,phospho- or carboxy-based cationic resin). The column can be washed witha buffer (such as chromatography buffer A/TMEG). The column can beeluted with a gradient of increasing NaCl concentration and fractionscan be collected and- assayed for the presence of AAV and/orcontaminants.

Other procedures can be used in place of or, preferably, in addition tothe above-described anion and cation exchange procedures, based oninter-molecular associations mediated by features other than charge asis known in the art. Such other procedures include intermolecularassociations based on ligand-receptor pairs (such as antibody-antigen orlectin-carbohydrate interactions), as well as separations based on otherattributes of the molecules, such as molecular sieving chromatographybased on size and/or shape. To take just a single example, the filtrateor partially purified AAV preparation may be loaded on a column thatcontains an AAV-specific antibody. This column can bind AAV. The columncan be rinsed with buffer to remove contaminating proteins, and theneluted with a gradient or step of increasing NaCl concentration andfractions can be collected. Alternatively, such a column can be elutedwith a buffer of different pH than that of the loading buffer.

The peaks of AAV and adenovirus can be identified in the fractions byinfectivity assays or by a nucleic acid hybridization or immunoassays.The peaks can be pooled, and the pool can be diluted or dialyzed ordiafiltered with a buffer (e.g. TMEG or equivalent) to reduce the saltconcentration.

This pool can be injected on a positively charged anion-exchange columnand/or a negatively charged cation-exchange column (such as thosereferred to above). The column can be washed with a buffer (such aschromatography buffer A/TMEG). The column can be eluted with a gradientof increasing NaCl concentration and fractions can be collected. Thepeaks of AAV and adenovirus can be identified in the fractions by aninfectivity assay or by a nucleic acid hybridization or immunoassay. Thepeaks can be pooled based on the results of any of these assays.

The pool of AAV-containing fractions eluted from an anion exchangecolumn as described above can be concentrated and purified by tangentialflow filtration (TFF), for example in a Filtron Ultrasette or MilliporePellicon unit. A membrane of suitable molecular weight cutoff (such as a100,00 or 300,000 cut-off), is typically composed of a polymer such asregenerated cellulose or polyethersulfone. The preparation is filteredthrough the membrane, and the product is retained. The retained materialcan be diafiltered using the membrane with successive washes of asuitable buffer such as Ringer's Balanced Salt Solution +5% glycerol.The final sample is highly enriched for the product and can be sterilefiltered through a 0.2μ filter and stored for use.

In the purification and concentration of AAV with tangential flowfiltration from post-anionic exchange column material, the 300,000molecular weight cut-off membrane has resulted in higher yields ofreplicative units than the 100,000 molecular weight cut-off membrane.

An additional step that can be employed for removal of adenovirus, ifdesired, involves treating the eluant pool with a heat inactivation step(as described herein) and then filtration (e.g. prior to subjecting thepreparation to TFF). However, we have found that the “anionexchange-to-TFF” procedure described above resulted in an AAVpreparation that was free of detectable adenovirus, and resulted inbetter yields of purified AAV.

In some embodiments, lysate or culture supernatant is subjected tofiltration (such as depth filtration) to clarify the lysate, followed byheat killing, followed by filtration (such as filtration using a 0.5 μmfilter) to further clarify the lysate, followed by cation exchangechromatography (using, for example, an HS resin), followed by nucleasedigestion, followed by anion exchange chromatography (using, forexample, a PI resin), followed by heparin sulfate chromatography,followed by gel filtration.

Illustrative production runs employing such techniques are describedbelow.

Altering the Growth Conditions of the AAV Producer Cells to EnhanceProduction

During the course of our production tests with AAV in various media andculture vessels, we typically monitored the cultures with respectvarious growth and/or metabolic parameters such as cell density,availability of glucose and amino acids, and the production of metabolicby-products such as ammonia and lactic acid. Such components can bereadily monitored using standard techniques such as HPLC and enzymaticassays, as described in the art.

As described in the Examples below, we discovered that certain aminoacids, particularly aspartate and glutamate, were rapidly depleted inboth batch and perfusion cultures. Indeed, in various batch andperfusion experiments, we have observed that from 90 to 99% of theavailable asp and glu is substantially eliminated after 24 to 48 hoursin such cultures. Since the levels of asp and glu appeared to besub-optimal in such media, we therefore provided additional amounts ofeither or both amino acid. Culture maintenance and optimizationtechniques such as these have been routinely applied in the context oflarge-scale bioproduction (see, e.g., Glacken, M. W., et al.,Biotechnology and Bioengineering, 28: 1376-1389, 1986; Glacken, M. W.,Bio/Technology 6: 1041-1050, 1988; Bibila, T. A., et al., Biotechnol.Prog., 10:87-96, 1994; and Borys, M. C., et al., Biotechnology andBioengineering, 43: 505-514, 1994).

To our surprise, replacement of these depleted amino acids resulted in asharp drop in AAV production. For example, in experiments describedbelow, supplementing the standard medium (DMEM) with additional asp andglu drove production efficiency down by more than an order of magnitude(from about 1800 DNase-resistant particles (DRP) per cell to about 140DRP per cell), although viability was slightly enhanced.

Another common component of media for the growth of mammalian producercells is a component of serum, such as fetal bovine serum (FBS), whichis typically included in media at a level of about 10%. As describedbelow, when the serum level for AAV production was increased (to 20%),AAV vector production dropped by more than 2-fold. In contrast, when thecells were subjected to increasingly lower levels of serum, AAV vectorproduction increased dramatically. For example, when serum levels werereduced to one-tenth of the normal starting levels (i.e. to 1%), vectorproduction increased by more than 4-fold.

Without wishing to be bound by theory, it now appears that stressing theproducer cells, either metabolically or by other means as describedbelow, can dramatically enhance the production of AAV vector. In someembodiments, the stress condition enhances production of rAAV vector (ascompared to production without imposing a stress condition(s)) at leastabout 2-fold, at least about 3-fold, at least about 5-fold, at leastabout 10-fold.

Stress can be effectively characterized, and tested, on the basis of thenegative effect of the stress condition or stress agent on cellulargrowth and/or metabolism. In effect, stress can be achieved by theintroduction of any condition or agent that inhibits cellular growthand/or metabolism, or by altering the level of a pre-existing conditionor agent such that it becomes sub-optimal with respect to cellulargrowth and/or metabolism. A large variety of such conditions are knownand/or apparent, including nutritional stress (one or more nutrientspresent at sub-optimal levels for growth and/or metabolism), temperaturestress (sub-optimal temperature, which may include growing the cells atlower or higher temperatures, or subjecting the cells to temporarytemperature shocks such as cold shock or heat shock), osmotic stress(sub-optimal osmotic level, which may be hypoösmotic or hyperosmotic),pH stress (sub-optimal pH which may be acidic or alkaline), aerationstress (e.g., sub-optimal levels of oxygen or gas exchange), mechanicalstress (e.g., shear stress as occurs in culture mixing), radiationstress, and toxic stress (presence of one or more chemicals or otheragents that inhibits growth and/or metabolism). With most if not all ofsuch agents and conditions, it is possible to subject the cells to thestress continuously, or temporarily. By way of illustration, in the caseof temperature stress, the cells can be grown at temperatures that areabove or below the optimum (typically the optimum is approximately thenormal body temperature of the animal from which the cells are derived),or the cells can be subjected to a temporary temperature shock, such asa cold shock or a heat shock. Presently preferred examples of suchstress conditions include: nutritional stresses, such as amino acid orserum limitation, the alteration of aeration levels and agitation, thealteration of osmotic levels (e.g. using non-metabolizable carbohydratessuch as sorbitol), and inclusion of chemical agents, such as saturatedaliphatic carboxylic acids (e.g., propionic, butyric, isobutryic,valeric and caproic acids and their salts with organic or inorganicbases), N,N′-diacylated diamines (such as pentamethylenebisacetamide,hexamethylenebisacetamide and heptamethylenebisacetamide), organicsulfur compounds (such as dimethylsulfoxide), and glucocorticoids (suchas hydrocortisone dexamethasone, prednisolone, aldosterone,triamcinolone and cortexolone). Other such agents include genotoxicagents such as chemical carcinogens, UV, heat shock, metabolicinhibitors of DNA synthesis (e.g., hydroxyurea, methotrexate,aphidicolin, drugs that affect topoisomerases (e.g., amsacrine,campthecin, etoposide and novobiocin).

As noted above, the producer cells can also be subjected to sub-lethalstress by altering pH. As exemplified below, we found that pH stressinduced by elevating medium pH not only increased AAV, but it alsocaused a dramatic shift in the relative proportions of AAV that werereleased into the culture medium. As further described below, thistechnique can thus be used to facilitate AAV purification as well asenhance production.

Illustrative procedures for optimizing the production of AAV byemploying various stress conditions are provided below; as are resultsdemonstrating that the application of a variety of different stressconditions can be used to effectively enhance AAV production levels.

Conditions Which Promote Release of rAAV Particles From Producer Cells

As described above with respect to pH, the present invention providesmethods for generating a population of recombinant adeno-associatedvirus (rAAV) particles, comprising the step of: a) incubating apopulation of producer cells in a cell culture medium under conditionsthat promote release of rAAV particles from the producer cells into theculture medium. The released rAAV particles may then be harvested fromthe cell culture medium, thereby obtaining a population of viralparticles.

Conditions which promote (or enhance) release of AAV (including rAAV)particles from a producer cell into the culture medium include, but arenot limited to pH of the culture medium; osmolality of the culturemedium; temperature; concentration of a given ion in the culture medium(which can affect conductivity); cell density; dissolved oxygenconcentration in the culture medium; glucose concentration in theculture medium; concentration of amino acids in the culture medium; andconditions which promote cell cycle synchronization. Any one or more ofthese parameters is maintained within a suitable range (i.e., a rangewhich promotes release) during which the cells release AAV particles.Although one parameter (i.e., condition) may be sufficient to promoterelease, a combination of two or more parameters can be simultaneouslymaintained, each within its own suitable range. Further, a suitablerange for one parameter may vary depending on whether any additionalparameter(s) used. If one parameter is held within a suitable range fora period of time, a second parameter can be maintained within a secondsuitable range for the same or a different period of time as the firstparameter. Alternatively, conditions may be employed serially. Forexample, control of pH may occur during one phase of growth, followed bycontrol of temperature.

It is well within the ability of one skilled in the art to vary theseparameters and to determine whether release of rAAV into the culturemedium is enhanced, relative to the amount of rAAV released whenproducer cells are not maintained under the given environmentalcondition(s). Enhanced (or increased) release of rAAV particles from aproducer cell can be measured by any of a number of methods known in theart, including, but not limited to, functional assays such as areplication center assay and an infectious center assay; immunologicalassays for cap gene products, such as ELISA; HPLC; and any of a numberof DNA detection methods (to detect the presence of viral DNA), such asslot blot.

Generally, any of these conditions may be monitored and controlled tothe extent necessary and/or desired using standard methods and equipmentknown in the art. The condition is measured and adjustments are made tomaintain or return the condition to its suitable level (i.e., a levelwhich promotes virus release). We have observed that failure toappropriately or adequately maintain culture conditions may result incessation of virus particle release, while other conditions, such asosmolality, need not be maintained at a specific level, but setting ofinitial culture condition with respect to this parameter(s) issufficient. For conditions that need to be monitored and adjusted, forexample, a bioreactor and/or media perfusion system may be used. Thesesystems are preferred, because they allow more careful control ofculture conditions. However, any system which allows sufficient controland adjustment of culture conditions to allow sufficient and/or desiredrelease of AAV particles is suitable. Other examples of controlmechanisms for providing the conditions are provided herein.

In one embodiment, producer cells are grown under pH conditions thatpromote viral particle release. Generally, pH of the culture medium ismaintained within a range of about 7.0 to about 8.5, preferably about7.4 to about 8.5, more preferably about 7.5 to about 8.0. Even morepreferably, and especially if pH is the only condition used to promoterelease, the pH is about 8.0. A bioreactor, for example, permits controlof pH to +/−0.05, and even more precise control is available (such as to+/−0.01), and can monitor pH every one to 3 minutes or even less. Insome embodiments, cells are grown under pH conditions whereby at leastabout 67% of total virus particles are found in culture supernatant. Inother embodiments, cells are grown under pH conditions such that atleast about 80%, at least about 82%, at least about 90%, at least about92%, at least about 95%, of virus particles are found in culturesupernatant. Preferably, the P/I, or particle to infectivity ratio, inculture supernatant is less than about 4,000, more preferably less thanabout 3,000, more preferably less than about 2,000, more preferably lessthan about 1,700, more preferably less than about 1,500. In someembodiments, cells are cultured at about pH 8 and are harvested on about96 hours from infection with helper virus (or introduction or initiationof helper virus function(s)). In other embodiments, cells are culturedat about pH 8 and are harvested on about 72 hours from infection withhelper virus (or introduction or initiation of helper virusfunction(s)). In other embodiments, cells are cultured at about pH 8 andare harvested on about 48 hours from infection with helper virus (orintroduction or initiation of helper virus function(s)).

In some embodiments a condition or conditions other than pH is used topromote virus release. For example, in other embodiments, temperature isused to promote virus release. Generally, the temperature of the culturemedium is maintained between about 30° C. to about 45° C., preferablybetween about 32° C. to about 42° C., more preferably about 35° C. toabout 40° C. Even more preferably, and especially if temperature is theonly condition to promote release, the temperature is about 37° to about39° C. A bioreactor for example can control a temperature to +/−0.5° C.,and can monitor as closely as about every 30 seconds.

In other embodiments, osmolality is used to promote virus release.Generally, the osmolarity of the culture medium is initiated and/ormaintained between about 100 mOsM to about 650 mOsM, preferably about150 mOsM to about 500 mOsM, preferably about 200 mOsM to about 400 mOsM,even more preferably about 300 mOsM (especially if osmolarity is theonly condition used to promote release). Osmolality, a term wellunderstood in the art, is defined as number of solute molecules per kgwater. Generally, compounds such as NaCl and other salts, mannitol,glucose, contribute to osmolality. Osmolality can be measured usingstandard techniques in the art, such as freezing point depression using,for example, an osmometer. As is understood in the art, use of an ionicsolute (such as Na or K) to adjust osmolality also can also affect otherparameters, such as conductivity. Accordingly, another condition whichmay be used to promote viral release is conductivity. Generally, theconductivity of the culture medium is initiated and/or maintained atleast about 5 mS, preferably at least about 10 mS, preferably at leastabout 15 mS (milliSiemens). In some embodiments, the lower limit ofconductivity is about any of the following: 5, 7, 10, 12, 15, 20 mS; andthe upper limit (selected independently of the lower limit) is about anyof the following: 7, 10, 12, 15, 20, 22, 25, 30 mS. Thus, for example,the conductivity (in mS) may range from about 5 to about 7, about 5 toabout 10, about 7 to about 10, about 10 to about 20, about 10 to about25. Preferably, the ion used to adjust conductivity is sodium (Na⁺).Conductivity can be measured using standard methods and devices in theart.

Other conditions that may be used in the methods of the presentinvention include, but are not limited to, any one or more of thefollowing: growth factors, such as insulin, EGF and FGF; glucoseconcentration; dissolved oxygen concentration; enriched media (e.g.,additional glucose and/or other nutrients such as vitamins and aminoacids). Glucose concentrations are generally between about 0.1 to about20 g/l; more preferably between about 0.5 to about 15 g/l; morepreferably between about 1 to about 10 g/l. Oxygen concentrations(typically measured by, for example, dissolved oxygen electrode or bloodgas analyzer) are generally between about 10% to about 200% relative toair, preferably between about 20% to about 100% relative to air,preferably about 30% to about 75% relative to air. Generally, the higherthe cell density, the more enriched the media. Media conditions may bemaintained and/or supplemented using techniques known in the art, suchas perfusion.

Producer cells are grown for a suitable period of time in order topromote release of virus into the media. Generally, cells may be grownfor about 24 hours, about 36 hours, about 48 hours, about 72 hours,about 4 days, about 5 days, about 6 days, about 7 days, about 8 days,about 9 days, up to about 10 days. After about 10 days (or sooner,depending on the culture conditions and the particular producer cellused), the level of production generally decreases significantly.Generally, time of culture is measured from the point of viralproduction. For example, in the case of AAV, viral production generallybegins upon supplying helper virus function in an appropriate producercell as described herein. Generally, cells are harvested about 48 toabout 100, preferably about 48 to about 96, , preferably about 72 toabout 96, preferably about 68 to about 72 hours after helper virusinfection (or after viral production begins). In the Examples, resultsare usually expressed number of days, e.g., “day 2”, “day 3”, etc. Thesedesignations generally indicate an additional day as measured frominfection with helper virus. That is, a result reported for “day 3”generally indicates that the result was obtained approximately 2 days,or 48 hours, from time of introduction of helper virus function(s).

As discussed above, any one or more, in any combination, of theconditions that promote release may be used. For example, cells may begrown under any one or more of the following conditions: (a) pH at about8.0; (b) temperature of about 39° C.; (c) about 300 mOsM; (d) enrichedmedia, for about 2 to 3 days. “Enriched media” generally mean enrichedin terms of additional inorganic salts (such as Mg², Ca⁺²) vitamins,and/or co-factors such that serum may be reduced or even eliminated. Ina preferred embodiment, cells are grown under the following conditions:(a) about 300 mOsm; (b) about pH 8.00; (c) about 39° C.; (d) and areharvested on about 96 hours from infection with helper virus. In apreferred embodiment, cells are grown under the following conditions:(a) about pH 8.00; (b) about 39° C.; and (c) and are harvested on about96 hours from infection with helper virus. In another embodiment, cellsare grown under the following conditions: (a) about 300 mOsm; (b) aboutpH 8.00; (c) about 39° C.; (d) and are harvested on about 72 hours frominfection with helper virus. In another embodiment, cells are grownunder the following conditions: (a) about pH 8.00; (b) about 39° C.; and(c) and are harvested on about 72 hours from infection with helpervirus. In another embodiment, cells are grown under the followingconditions: (a) about 300 mOsm; (b) about pH 8.00; (c) about 39° C.; (d)and are harvested on about 48 hours from infection with helper virus. Inanother embodiment, cells are grown under the following conditions: (a)about pH 8.00; (b) about 39° C.; and (c) and are harvested on about 48hours from infection with helper virus. In another embodiment, cells aregrown under the following conditions: (a) about pH 8.00; (b)conductivity of at least about 10 mS, or in alternative embodiments,conductivity of about 15 mS, preferably about 17 mS. In anotherembodiment, cells are grown under the following conditions: (a) about pH7.2 to about 7.4; (b) conductivity of at least about 10 mS, oralternative embodiments, conductivity of about 15 mS, preferably about17 mS.

In some embodiments, pH is maintained at about 8.0, and the culture isgrown at a temperature of about 39° C. In some embodiments, pH ismaintained at about 8.0 and the osmolality (at least the initialosmolality) is about 300 mOsm. In some embodiments, pH is maintained atabout 8.0, the osmolality (at least the initial osmolality) is about 300mOsm, and the culture is grown at a temperature of about 39° C. In someembodiments, an ionic salt such as a sodium salt is used to adjustand/or maintain osmolality and/or pH.

In some embodiments, cells are synchronized. This may be accomplished,for example, by subjecting the cells to stress conditions, particularlybefore addition of helper virus function(s). Synchronization maycontribute to overall productivity. Possible forms of stress include,but are not limited to, a nutritional stress, an osmotic stress, a pHstress, a temperature stress, an aerobic stress, a mechanical stress, aradiational stress and a toxic stress. A non-limiting example by whichnutritional stress is imposed is by culturing the producer cells in amedium that is deficient in one or more amino acids.

It is also understood that, the invention also includes methods oftreating producer cells (i.e., intact producer cells) with an agent orcondition that promotes virus release, for example, treatment with anagent which permeabilizes a cell, such as an ionophore or a toxin (suchas a bacterial toxin), and/or osmotic shock. Agents and conditions whichpermeabilize cells are known in the art. Examples include, but are notlimited to, detergents such as saponin, igitonin, Triton X-100, sodiumdodecyl sulfate (SDS), C12E8, sodium dodecyl sulfate, sodium cholate andsodium deoxycholate; small inorganic and organic molecules such asglycyl-L-phenylalanine-beta-naphthylamide, Cu2+ ions or methylamin,nystatin, harzianins HC; peptides and proteins such as magainin,rotavirus capsid protein VP5*, amphotericin B, streptolysin O, defensinA, cryptdins 2 and 3, and C5b-9; physical conditions such aselectroporation and cell scraping; phospholipids such as lysolecithin(lysophosphatidylcholine). For these embodiments, the producer cells ofa culture population generally retain their integrity, i.e., are notlysed (although, as in any cell culture population, some cells may belysed). Generally, less than about any of the following percentage ofcells are lysed: 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 12%, 10%, 8%,5%, 3%, 2%, 1%. Alternatively, generally about at least any of thefollowing percentage of cellular contents are retained in cells (i.e.,retained in the cell membrane): 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%.

Examples of suitable culture media are described in the Examples.

Harvesting and Purifying Released Viral Particles

Producer cells may be cultured in suspension or attached to a suitablesurface. Methods of suspension or fixed cultures are known in the art.Upon generation of a population of released viral particles, thereleased viral particles may be harvested and/or purified for furtheruse. As discussed in more detail herein, virus particles in culturemedia are separated from producer cells using methods known in the art,such as centrifugation or filtration (such as tangential flow filtrationin a hollow-fiber membrane). Preferably, one or more additionalpurification steps are performed after separating producer cells fromthe culture medium. Examples of such steps include, but are not limitedto, concentration using suitable filters or ion exchange chromatography.Various production and purification methods are described in WO 99/11764(PCT/US98/18600) (Targeted Genetics Corporation). Any purificationstep(s) described herein may be applied to culture supernatant, as wellas those known in the art (in any combination).

In some embodiments, culture supernatant (after cells are removed) issubjected to opposing ionic chromatography, as described above. In someembodiments anion-exchange chromatography is followed by cation-exchangechromatography. In other embodiments, cation-exchange is followed byanion-exchange chromatography. In some embodiments, culture supernatantis subjected to chromatography on heparin sulphate, preferably aftertreatment of opposing ionic chromatography, more preferably aftertreatment of culture supernatant on cation exchange chromatographyfollowed by anion exchange chromatography. In some embodiments, thesupernatant is subjected to cation exchange chromatography followed byanion exchange chromatography followed by cation exchangechromatography. Preferably, heparin sulfate is used in at least one(preferably the last) cation exchange chromatography. These techniquesare described in more detailed below.

By way of illustration, culture supernatant, or a preparation which hasbeen eluted from an anion-exchange or cation-exchange column and/orconcentrated by tangential flow filtration can be purified by binding toa column comprising heparin sulphate which serves as a cationic exchangeresin. The AAV can then be eluted from such a column using a buffercontaining a salt (eg, a linear gradient of NaCl). For example, AAVobtained from pooled fractions from anion-exchange chromatography columncan be concentrated and diafiltered into TMEG plus 100 mM NaCl using a300K tangential flow filtration membrane. This concentrate may beinjected on a one ml heparin sulphate column (Pharmacia “Hi-TrapHeparin” column), and eluted using a linear gradient of NaCl.

Use of rAAV for Gene Therapy

Embodied in this invention are vector compositions comprisingpolynucleotides with a therapeutically relevant genetic sequence. AAVviral vectors of this invention can be used for administration to anindividual for purposes of gene therapy. Suitable diseases for genetherapy include but are not limited to those induced by viral,bacterial, or parasitic infections, various malignancies andhyperproliferative conditions, autoimmune conditions, and congenitaldeficiencies.

Gene therapy can be conducted to enhance the level of expression of aparticular protein either within or secreted by the cell. Vectors ofthis invention may be used to genetically alter cells either for genemarking, replacement of a missing or defective gene, or insertion of atherapeutic gene. Alternatively, a polynucleotide may be provided to thecell that decreases the level of expression. This may be used for thesuppression of an undesirable phenotype, such as the product of a geneamplified or overexpressed during the course of a malignancy, or a geneintroduced or overexpressed during the course of a microbial infection.Expression levels may be decreased by supplying a therapeuticpolynucleotide comprising a sequence capable, for example, of forming astable hybrid with either the target gene or RNA transcript (antisensetherapy), capable of acting as a ribozyme to cleave the relevant mRNA,or capable of acting as a decoy for a product of the target gene.

Of particular interest is the correction of the genetic defect of cysticfibrosis, by supplying a properly functioning cystic fibrosistransmembrane conductance regulator (CFTR) to the airway epithelium.Afione et al. (J. Virol. 70:3235, 1996) and Conrad et. al. (GeneTherapy: in press, 1996) have shown stable in vivo CFTR gene transfer tothe primate lung using single-dose AAV vectors. There are a variety ofCFTR polypeptides that are capable of reconstructing CFTR functionaldeficiencies in cells derived from cystic fibrosis patients. Rich etal., Science, 253: 205 (1991) described a CFTR derivative missing aminoacid residues 708-835, that was capable of transporting chloride andcapable of correcting a naturally occurring CFTR defect. Egan et al.,Nature, 358:581 (1992) described another CFTR derivative (comprisingabout 25 amino acids from an unrelated protein followed by the sequenceof native CFTR beginning at residue 119) that was also capable ofrestoring electrophysiological characteristics of normal CFTR Arispe etal., Proc. Natl. Acad. Sci. USA 89: 1539 (1992) showed that a CFTRfragment comprising residues 433-586 was sufficient to reconstitute acorrect chloride channel in lipid bilayers. Sheppard et al., Cell 76:1091 (1994) showed that a CFTR polypeptide truncated at residue 836 toabout half its length was still capable of building a regulated chloridechannel. Thus, AAV vectors with encoding sequences for native CFTRprotein, and mutants and fragments thereof, are all preferredembodiments of this invention.

Also of particular interest is the correction of the p53 tumorsuppressor gene, locally defective in certain tumor types, by supplyinga properly functioning p53 gene to the tumor site (Huyghe et al., HumanGene Therapy 6:1403, 1995).

Compositions of this invention may be used in vivo as well as ex vivo.In vivo gene therapy comprises administering the vectors of thisinvention directly to a subject. Pharmaceutical compositions can besupplied as liquid solutions or suspensions, as emulsions, or as solidforms suitable for dissolution or suspension in liquid prior to use. Foradministration into the respiratory tract, a preferred mode ofadministration is by aerosol, using a composition that provides either asolid or liquid aerosol when used with an appropriate aerosolizerdevice. Another preferred mode of administration into the respiratorytract is using a flexible fiberoptic bronchoscope to instill thevectors. Typically, the viral vectors are in a pharmaceutically suitablepyrogen-free buffer such as Ringer's balanced salt solution (pH 7.4).Although not required, pharmaceutical compositions may optionally besupplied in unit dosage form suitable for administration of a preciseamount.

An effective amount of virus is administered, depending on theobjectives of treatment. An effective amount may be given in single ordivided doses. Where a low percentage of transduction can cure a geneticdeficiency, then the objective of treatment is generally to meet orexceed this level of transduction. In some instances, this level oftransduction can be achieved by transduction of only about 1 to 5% ofthe target cells, but is more typically 20% of the cells of the desiredtissue type, usually at least about 50%, preferably at least about 80%,more preferably at least about 95%, and even more preferably at leastabout 99% of the cells of the desired tissue type. As a guide, thenumber of vector particles present in a single dose given bybronchoscopy will generally be at least about 1×10⁸, and is moretypically 5×10⁸, 1×10¹⁰, and on some occasions 1×10¹¹ particles,including both DNAse resistant and DNAse susceptible particles. In termsof DNAse resistant particles, the dose will generally be between 1×10⁶and 1×10¹⁴ particles, more generally between about 1×10⁸ and 1×10 ¹²particles. The treatment can be repeated as often as every two or threeweeks, as required, although treatment once in 180 days may besufficient.

The effectiveness of the genetic alteration can be monitored by severalcriteria. Samples removed by biopsy or surgical excision may be analyzedby in situ hybridization, PCR amplification using vector-specificprobes, RNAse protection, immunohistology, or immunofluorescent cellcounting. When the vector is administered by bronchoscopy, lung functiontests may be performed, and bronchial lavage may be assessed for thepresence of inflammatory cytokines. The treated subject may also bemonitored for clinical features, and to determine whether the cellsexpress the function intended to be conveyed by the therapeuticpolynucleotide.

The decision of whether to use in vivo or ex vivo therapy, and theselection of a particular composition, dose, and route of administrationwill depend on a number of different factors, including but not limitedto features of the condition and the subject being treated. Theassessment of such features and the design of an appropriate therapeuticregimen is ultimately the responsibility of the prescribing physician.

The foregoing description provides, inter alia, methods for generatinghigh titer preparations of recombinant AAV vectors that aresubstantially free of helper virus (e.g. adenovirus) and cellularproteins. It is understood that variations may be applied to thesemethods by those of skill in this art without departing from the spiritof this invention.

The examples presented below are provided as a further guide to apractitioner of ordinary skill in the art, and are not meant to belimiting in any way.

EXAMPLES Example 1 Illustrative Production of Recombinant AAV VectorUsing a Wild-Type Helper Virus (AD5) and a Temperature-Sensitive HelperVirus (AD TS149)

This example illustrates the use of a wild-type helper virus (Ad5) and atemperature-sensitive helper virus (Ad ts149) to provide helperfunctions for the replication of a recombinant AAV vector particlecomprising a model therapeutic gene.

The ptgAAVCF plasmid consists of the left hand AAV2 ITR; a full lengthcystic fibrosis transmembrane regulator cDNA; a syntheticpolyadenylation sequence based on the mouse β-globin polyadenylationsequence; AAV2 sequences downstream of the cap coding sequences; and theright-hand AAV2 ITR in a pBR322 plasmid backbone (Afione et al,, 1996).The pGEM-RS5 packaging plasmid was derived from the pHIVrep plasmid(Antoni et al., 1991) and consists of the U3 and R regions from theHIV-1 LTR; the rep and cap regions from AAV2 including the p19 and p40promoters; pBR322 and pGEM plasmid sequences for bacterial replicationand selection; and a small region of human Alu repetititve cellular DNAupstream of the HIV LTR.

Adenovirus type 5 was grown from a stock obtained from the American TypeCulture Collection (Rockville, Md.). Ad5ts149 (Ensinger et al., J.Virol. 10:328, 1972) was obtained from Harold S. Ginsberg.

Working stocks of Ad5 and Ad5ts149 (ts149) were produced at 37° C. and32° C.; respectively, by infecting 293-1 cells at a multiplicity ofinfection (MOI) of 5 and 1; respectively. After 4 hours the cultureswere refed with fresh medium and incubated at 37° C. in a humidified 10%CO₂ incubator. After seventy-two hours, cells were removed, pelleted at1000 g at 15° C. and resuspended in PBS containing 0.1 g/L of MgCl₂ and0.1 g/L CaCl₂. The cell suspension was then frozen and thawed threetimes, sheared three times through an 18 gauge needle and clarified bycentrifugation at 1000 g at 15° C. The clarified lysate was then treatedwith DNase I at a final concentration of 2 mg/ml for 30 minutes at 37°C. The treated lysate was layered on a discontinuous step gradient ofCsCl in water comprising 4.0 ml of CsCl (1.25 g/cm³) layered over 2.0 mlof CsCl (1.40 g/cm³) in water and centrifuged at 35,000 RPM for 1 to 2hours in a Beckman SW41 rotor. The adenovirus band from each tube wasremoved, pooled and diluted in 1.35 g/cm³ CsCl in water and centrifugedovernight at 35,000 RPM in a Beckman SW55 rotor. The adenovirus band waspooled, adjusted to 10% glycerol and dialyzed extensively against 10 mMTris pH 7.5 buffer supplemented with 1.0 mM MgCl₂ and 10% glycerol.293-1 cells (ATCC CRL 1573) were maintained in T-flasks in a humidified10% CO₂ incubator in DMEM high glucose medium (JRH) supplemented with10% fetal bovine serum (FBS, Hyclone). For this example, the 293-1 cellswere inoculated at 4.4×10⁴ cells/cm² in tissue culture flasks with DMEMsupplemented with 10% FBS and 2.0 mM L-glutamine, and incubated fortwenty-four hours at 37° C. in a humidified 10% CO₂ incubator.

The cells (about 10⁷ cells per flask) were then infected with workingstocks of either Ad5 or ts149 for 1 hour at a MOI of 5, followed bytransient transfection of vector and packaging plasmids. Transientco-transfection of ptgAAVCF vector plasmid and pGEM-RS5 helper plasmidwas performed using LIPOFECTAMINE™ (Gibco). In that process, 37.5 μg ofeach plasmid along with 150 μl LIPOFECTAMINE™ were mixed and diluted in4.75 ml of serum-free MEM. The adenovirus inoculum was removed and theplasmid-LIPOFECTAMINE™ mixture was added to the cells and incubated forfour hours in a 5% CO₂ incubator at the appropriate temperature. Theplasmid-LIPOFECTAMINE™ mixture was removed from the culture after fourhours and replaced with fresh medium.

Cells infected with wild-type virus were cultured at 37° C. and cellsinfected with Adts149 were incubated at 39.5° C. After 72 hours, thecells were harvested, pelleted and resuspended in 10 mM Tris pH 7.5. Thesuspension was then lysed by sonication in a ice-water bath using aBranson cup-horn sonicator utilizing four 15 second pulses and assayedfor rAAVCF and adenovirus production.

Example 2 Quantitation of RAAV and Adenovirus Titers in VectorPreparations

Cell lysates from the preceding example were assayed for production ofrAAVCF vector by C37 replication assay and analyzed for adenovirusproduction by slot-blot hybridization.

HeLa C37 was constructed to allow inducible expression of AAV Repproteins for rAAV vector replication. Briefly, an AAV Rep/Cap expressioncassette consisting of the mouse metallothionein I promoter, AAV2 repand cap genes and AAV transcription termination site was constructed.Also included in the plasmid was a neomycin resistance gene under thecontrol of the SV40 early promoter, SV40 small T intron and the SV40polyadenylation signal. HeLa cells were transfected with the plasmid andclones were selected in G418. A panel of clones was screened by a rAAVvector amplification assay. One clone, C37, demonstrated consistent anddose dependent amplification of rAAV vector following transduction andadenovirus infection.

Detection of replicating vector is accomplished by DNA isolationfollowed by hybridization to a CFTR probe. In detail, HeLa C37 cellswere inoculated at 4.4×10⁴ cells/cm² in tissue culture flasks with DMEMsupplemented with 10% FBS and 2.0 mM L-glutamine and incubated fortwenty-four hours at 37° C. in a humidified incubator at 5% CO₂. Thecells were then inoculated with adenovirus (MOI=5) and dilutions ofrAAVCF sample for 72 hours. Cells were harvested by scraping andprepared for Southern blot analysis. Total cellular DNA was prepared,digested with EcoRI, electrophoresed on a 1% agarose gel, transferred toa nylon 66 membrane followed by hybridization with a ³²P-labeled humanCFTR cDNA restriction fragment. This probe detects an approximately 1.5kb fragment from the AAVCF vector (corresponding to the predicted 1.488kb EcoRI fragment). Vector replication was quantitated relative to anendogenous genomic CFTR band and is expressed as replication units. Onereplication unit (RU) is defined as a signal intensity equivalent tothat of the endogenous genomic CFTR band which is approximately 1.8 kb.In some experiments, linear regression of serially diluted known vectorstandards was used to extrapolate and calculate vector concentration insamples.

The adenovirus DNA slot blot assay was conducted as follows. Aliquots ofsamples were denatured in 0.4M NaOH, 10 mM EDTA with 1.0 μg/ml salmonsperm DNA at 65° C. Samples and adenovirus standards were diluted andfiltered onto nylon membranes using a slot blot manifold and washed with0.4M NaOH. The filter was hybridized with a ³²P-labeled probecorresponding to the adenovirus E1A gene sequence. The entire Ad5 genomeis available on Genbank at accession number X02996. We used a 1 kbSspI-XbaI fragment (corresponding to nucleotides 339-1339) and analyzedthe blots on a phosphorimager (Molecular Dynamics). One genomeequivalent was considered to be equivalent to one adenovirus particle.

FIG. 1 shows the results of the replication assay for rAAVCF vector inlysates prepared with Ad5 or ts149 at permissive (37° C.) andnon-permissive temperatures (39.5° C.). Production of recombinant vectorwas supported by ts149 at 39.5° C. but productivity was approximately 2to 3 fold less than Ad5.

FIG. 2 shows the results of the slot blot assay to determine thequantity of adenovirus. Production of adenovirus genomes was reduced 34logs by use of the temperature sensitive mutant as compared towild-type.

Example 3 Optimization of Helper Function to Improve RAAV Production

This example illustrates various attempts to improve the level of rAAVobtained when using temperature-sensitive helper virus. Increasinginfection levels of the helper virus was unhelpful, but adjusting thekinetics was surprisingly effective.

The effects of increasing multiplicity of infection on vector productionwas evaluated first. 293-1 cells were infected with either Ad5 at a MOIof 5 or ts149 at various MOI, followed by transient co-transfection withvector and packaging plasmids. After 72 hours, the cells were lysed andassayed for production of rAAVCF vector by C37 vector replication assayand analyzed for adenovirus production by slot-blot hybridization. Anadditional 96 hour time point was collected for cells infected withts149 at a MOI of 5.

FIG. 3 shows the results of the rAAVCF replication assay conducted oncell lysates prepared with ts149 at various MOI. Increasing the MOI ofts149 did not restore vector productivity to levels observed with Ads(as shown by the intensity of the 1.4 kb hybridization band). However, ahigher level of vector production was observed at the 96 hour timepoint. The concentration of ts149 in the lysate detected by slot blotanalysis increased with increasing MOI, but were still 3 to 4 logs lowercompared with Ad5.

Following the observation of increased vector productivity with ts149 at96 hours in the previous experiment, a time course and productionkinetic study was performed. 293-1 cells were infected with either Ad5or ts149 at a MOI of 5 followed by transient co-transfection with vectorand packaging plasmids. Cells infected with Ad5 and ts149 were culturedat 37° C. and 39.5° C.; respectively, for six days. Lysates from days 3,4, 5 and 6 were assayed for vector production by vector replicationassay and analyzed for adenovirus by slot-blot hybridization.

FIG. 4 illustrates the kinetics of vector production. Solid barsrepresent lysates produced using wild-type Ad5 as helper; hatched barsrepresent lysates produced using ts149 as helper. Maximal vectorproduction when using Ad5 was ˜2.0×10⁶ RU/ml, peaking at day 4. At thistime point, the vector production obtained using ts149 was less than˜0.3×10⁶ RU/ml. On day 5, however, there was a dramatic alteration inthe relative efficacy of the two helper viruses. Vector productionsupported by Ad5 fell to below 0.3×10 RU/ml. In contrast, vectorproduction supported by ts149 jumped to over 2×10⁶ RU/ml. Adenovirusgenome levels observed when using ts149 were significantly lower thanwith Ad5.

Example 4 Development of Suspension Cultures for Producing Helper Virus

The preceding example shows that the levels of temperature-sensitiveadenovirus produced by conventional culture techniques is low. Thislimits the ability to use temperature-sensitive adenovirus as helpers inproduction of AAV vectors. The present example provides an improvedmethod that allows for the production of temperature-sensitiveadenovirus in much higher amounts. Central to the improvement is the useof host cells grown in suspension culture.

293 N3S and HeLa S3 are suspension variants of the 293-1 human embryonickidney and HeLa human epitheloid carcinoma cell lines; respectively.Suspension cultivation was performed in 500 ml spinner flasks (Bellco)with working volumes of 250 to 300 ml. HeLa S3 (ATCC 2.2-CCL) cells weremaintained in DMEM/F-12 with 15 mM HEPES supplemented with 7.5% FBS and2.0 mM L-glutamine. 293-1 N3S (Microbix Biosystems Inc.) were passagedin Joklik MEM supplemented with 7.5% FBS and 2.0 mM L-glutamine.Spinner-flasks were agitated at 50-65 RPM.

Growth performance was assessed in the following experiment. 293 N3S andHeLa S3 were serially passaged in suspension in replicate 500 ml spinnerflasks and cell growth and viability was monitored. Flasks wereinoculated at cell densities of 2 to 5×10⁵ cells/ml and then culturedfor 2 to 3 days. To control for seeding density differences, populationdoubling levels (PDLs) were compared for replicate cultures. The averagePDL was 2.0±0.49 (mean±SD.) and 1.1±0.62 for HeLa S3 and 293 N3S;respectively (n=14). Higher cell doublings were consistently observedwith the HeLa S3 cells. Cell morphology in suspension was dramaticallydifferent for the two lines. HeLa cells grew as single cells or smallaggregates. In contrast, 293 N3S cells formed large aggregates of 50 to100 cells each. Significant numbers of non-viable cells were observed inthe center of the large clumps. Stocks were subcultivated bycentrifugation followed by gentle disruption with a pipette releasingthe non viable cells from the aggregates. Initial culture viabilities of293 N3S were consistently lower compared to HeLa S3.

Based on cell growth, viability and morphology in suspension, the HeLaS3 cell line was selected for further process development. Growth andviability at permissive temperatures were evaluated. HeLa S3 cells wereseeded into 500 ml spinner flasks at 5×10 cells/ml, and monitored dailyfor seven days.

FIG. 5 shows the viable cell density (VCD) of HeLa S3 cells, grown at32° C. (squares) and 37° C. (circles). Bars about the 32° C. time pointsindicate the range of values observed in replicate 500 ml spinnerflasks. Cells grown at 37° C. peaked at 2.5×10⁶ cells/ml on day 5,whereas cells grown at 32° C. peaked at 2×10⁶ cells/ml on day 6.Viability (determined by trypan blue exclusion) was at least about 90%throughout.

Tangential flow or cross flow filtration is a versatile technique for awide variety of large scale biopharmaceutical applications includingconcentration or removal of cells, concentration of macromolecules andmedia/buffer exchange. Tangential flow processing is required forconcentrating cells for infection and for harvesting infected cells atlarge scale.

The effect of laminar shear on cell viability in tangential flowfiltration was evaluated by concentrating and diafiltering the HeLa S3cells. HeLa S3 cells were inoculated at a density of 4×10⁵ cells/ml inthree liter Applikon bioreactors and cultured to 2×10 cells/ml inDMEM/F-12 with 15 mM HEPES (JRH) supplemented with 7.5% FBS, 2.0 mMglutamine, 1×MEM amino acids, 1×MEM non-essential amino acids, 0.1%Pluronic polyol F-68 and 2 g/L glucose. Bioreactor working volume wastwo liters. Dissolved oxygen, pH, temperature and agitation werecontrolled at 60% (relative to air saturation), 7.2, 37° C. and 100 rpm;respectively, using the FERMCON™ (Scius Corporation) controller system.

Tangential flow filtration experiments were performed with mixedcellulose ester hollow fiber membranes (Microgon). Pore size and surfacearea was 0.2μ and 725 cm²; respectively. A 0.2μ filter was selected toretain cells while allowing passage of spent media. Cells were pumped(Cole Palmer) through the inside diameter of the hollow fibers.Recirculation rates were adjusted to provide average wall shear rates of750 and 1500 sec⁻¹. Once the crossflow was established, permeate flowcontrol of 30 and 90 m/min; respectively, was achieved by a pump (ColePalmer) located on the permeate line. During cell concentration,permeate withdrawal continued until the desired fold concentration wasachieved. During diafiltration, media feed entering the bioreactor wasactivated until the desired fold medium exchange was achieved. Viablecells were counted before and after each treatment.

FIG. 6 shows the growth curves of HeLa S3 cells before and aftertangential flow processing in an exemplary experiment. Two liters ofcells were cultured in 3 liter bioreactors. On day 3 (arrow), cells wereconcentrated seven fold from the 2-liter working volume, diafilteredagainst six volumes of growth medium and brought up to the originalworking volume. The results show that the cells were not damaged by wallshear of 750 sec⁻¹ (squares) and 1500 sec⁻¹ (circles), and continued togrow to high cell densities.

Suspension cultures of HeLa S3 cells were then tested as host cells forts149 production, or their ability in 300 ml suspension culture wasinvestigated. HeLa S3 cells from 300 ml suspension culture (1×10⁶cells/ml) were centrifuged, concentrated and infected with ts149(MOI=3). After 1 hour, the culture was transferred to a spinner-flask,resuspended in media and cultured for seven days at 32° C. The HeLa S3cells continued to grow from about 1×10⁶ cells/ml at the time ofinfection to about 2×10⁶ cells/ml by day 5. Viability decreased to ˜60%on day 7.

FIG. 7 shows the production of ts149 by HeLa S3 cell cultures. Theculture was sampled daily, and lysates were prepared by freeze-thaw foranalysis of virus production by the adenovirus infectivity assay. Virusproduction reached ˜4.5×10⁷ IU/ml of culture by about day 3-5. On day 7,the cells were collected by centrifugation, resuspended in TMEG bufferand lysed by microfluidization (MF). The infectious titer of themicrofluidized lysate was comparable to those of the freeze-thaw lysatesample indicating recovery by microfluidization was comparable tofreeze-thaw methods.

Example 5 Improved Purification Method for the Production of aTemperature-Sensitive Helper Virus (AD TS149)

Purification using CsCl gradients is burdensome for large scaleproduction. This example illustrates the purification of ts149 by ionexchange chromatography.

Chromatography was performed on a Perseptive Biosystem BIOCAD™chromatography workstation. The resin used was a polyethyleneimine (PI)weak anion exchanger (POROS™ 50 PI). The column was equilibrated withTMEG (50 mM Tris, pH 8.0, 5 mM MgCl₂, 1 mM EDTA, 5% glycerol).Chromatography was monitored on-line for pH, conductivity and opticaldensity at 280 nm.

Suspension HeLa S3 infected with ts149 at a MOI of 2 was harvested andcentrifuged. The pellet was resuspended in TMEG and lysed by cavitationat 3000 PSI using a microfluidizer (Microfluidics). Lysate was clarifiedby filtration through a 5μ syringe filter (Millex SV) followed by a0.45μ syringe filter (Acrodisc). Clarified lysate was loaded onto a 1.6ml POROS™ 50 PI anion exchange column run at 1 ml/min. The column waswashed with 10 column volumes of TMEG with 900 mM NaCl, and the ts149was eluted with a linear gradient from 900 to 1300 mM NaCl. Fractions of0.5 ml were collected and assayed by infectivity assay and slot blot forthe presence of adenovirus.

FIG. 8 shows the results of the infectivity assay conducted onconsecutive column fractions. The majority of the infectious adenoviruswas found in fractions 26 to 28, coincident to the peak of absorbanceeluting at about 100 ms at approximately 25 minutes. The ts149 elutedjust prior to the large peak at higher salt concentration. Infectivityand slot blot assays conducted in parallel confirmed particles andinfectious virus were in the same peak fractions.

Lysate and PI peak fractions were also assayed for total protein by theBradford method. Protein concentration was 1.8 mg/ml in the lysate andless than 30.0 μg/ml in the PI pool. The virions were separated from themajority of cellular protein in a single step and eluted as a singlepeak. The virions showed very high affinity for the PI matrix, asevidenced by the relatively high salt concentration required to elutethem from the column.

Large-scale production method for temperature-sensitive helper virus canincorporate all the improvements described in these examples. In oneillustration, virus production would comprise the following steps:

-   -   Cell culture in suspension bioreactor    -   Concentration/Medium exchange    -   Infection with helper virus    -   Virus production    -   Harvest: Concentration/Diafiltration    -   Lysis by microfluidization    -   PI ion-exchange chromatography    -   Concentration/Diafiltration    -   Sterile filtration

This type of approach is inherently scalable and amenable to currentGood Manufacturing Practices.

Additional exemplary illustrations of such techniques are providedbelow.

Example 6 Comparison of First and Second Generation Processes for HelperVirus Production

A. Illustrative First Generation Helper Virus Production and Processing

In an exemplary “first generation” process for helper virus production,mammalian cells were grown in 40 T225 flasks, and then infected with Ad5at an MOI of about 1. After incubating, the cells were harvested bycentrifugation, and lysed by freeze-thawing and passage through aneedle. The lysate was subjected to treatment with DNase I and then runon a step CsCl gradient and isopycnic gradient. Purified material wasdialyzed and sterile filtered.

Using this first generation process, we obtained approximately 1×10¹²particles (or approx. 1×10¹¹ infectious units) from 4×10⁸ cells.

B. Illustrative Second Generation Helper Virus Production and Processing

In an exemplary “second generation” process for helper virus production,mammalian cells (HeLa S3) were grown in 10 liter bioreactors, and theninfected with Ad5 (from ATCC, subsequently plaque-purified on 293 cells,serially expanded on HeLa S3 cells and double purified by CsCl gradientcentrifugation) at an MOI of about 1. After incubating, the cells wereconcentrated and harvested by diafiltration, and lysed bymicrofluidization. The lysate was subjected to treatment with Benzonase(nuclease) and then filtered. The filtrate was then run on an anionexchange column (PI)concentrated and diafiltered, and finally sterilefiltered.

Using this second generation process, we obtained approximately 1×10¹⁴particles (or approx. 5×10¹² infectious units) from 1×10¹⁰ cells.

FIG. 9 illustrates the results of the downstream processing of helpervirus using anion exchange chromatography as described above. Bars:Viral activity measured in an infectivity assay; Solid line: A₂₈₀;Dotted line: buffer conductivity (mS).

As is apparent from comparing the fractionation of viral activity versusA280 absorbance, these processing procedures resulted in a substantialseparation of the helper virus from the bulk of contaminating materialswhich would be expected to contain cellular proteins and nucleic acids.

Example 7 Comparison of First and Second Generation Processes forProduction of Recombinant AAV Vectors

A. Illustrative First Generation rAAV Production and Processing

In an exemplary “first generation” process for rAAV vector production,mammalian cells were grown in 40 T225 flasks, and then infected with Ad5at an MOI of about 5. After incubating, the cells were harvested bycentrifugation, and lysed by sonication. The lysate was subjected totreatment with DNase I and then run on a series of two CsCl gradients.Purified material was dialyzed and sterile filtered. Using this firstgeneration process, we obtained approximately 5×10⁶ replicative unitsRUs from 4×10⁸ cells.

B. Illustrative Second Generation rAAV Production and Processing

In an exemplary “second generation” process for rAAV vector production,mammalian cells were grown in 10 liter bioreactors, and then infectedwith Ad5 at an MOI of about 5. After incubating, the cells wereconcentrated and harvested by diafiltration, and lysed bymicrofluidization. The lysate was subjected to treatment with Benzonase(nuclease) and then filtered. The filtrate was then run on anionexchange column, followed by a cation exchange column. Eluant fractionscontaining AAV were pooled, concentrated and diafiltered, and finallysterile filtered. This second generation process is expected to yieldgreater than 1×10¹¹ replicative units RUs from 1×10¹⁰ cells.

FIG. 10 show the results of sequential fractionation on ion exchangecolumns: first, on an anion exchange matrix (upper panel), and then on acation exchange matrix (lower panel). Bars: Viral activity measured inan infectivity assay for either Adenovirus or AAV; Solid line: A₂₈₀ (ameasure of total protein); Dotted line: buffer conductivity (mS). As isapparent from the analyzed fractions, it is possible to obtain extremelyhigh levels of separation between AAV and Adenovirus, as well as betweenAAV and A280-absorbing material (largely proteins) using the techniquesof the present invention. In particular, the results revealed that AAVvectors can be retained on both anionic and cationic exchange columns,and that the differential elution of AAV using both anionic and cationicexchange resulted in dramatically enhanced ability to separate AAV fromall of the major contaminants of interest (including Adenovirus as wellas cellular proteins).

In another exemplary second generation process for rAAV vectorproduction, the filtrate was prepared as described above and was thenrun on an anion exchange column, followed by pooling of eluant fractionscontaining AAV, and then subjecting the pooled anion exchange eluants totangential flow filtration (TFF). As described below, this anionexchange to TFF procedure was found to result in a highly concentratedand purified preparation of AAV.

Detailed analysis of AAV obtained using such second generationtechnology, using techniques as described above and in the art(including infectivity assays, slot blot analyses and SDS gelelectrophoresis) provided further confirmation that the material was ofhigh quality and substantially free of contaminating adenovirusparticles (and adenovirus protein and DNA), and also substantially freeof contaminating cellular proteins and DNA. SDS gels revealed thepresence of bands corresponding to VP1, VP2 and VP3 (i.e. the AAV capsidproteins). No other bands were visible after Coomassie staining. Thesedata are consistent with the results of the column fractionationanalyses as depicted in FIGS. 10-11.

As an illustrative anion exchange to TFF procedure, the following is anexamplary purification and concentration process starting with one literof pooled fractions from anion exchange chromatography. If desired (asnoted above), this pool can be subjected to heat inactivation followedby a filtration step (e.g., using a 0.22 μM filter). For tangential flowfiltration (TFF), we employed a sanitized Pellicon XL system equippedwith a 300,000 molecular weight cut-off membrane which was operated at40/0 for inlet and outlet pressures. One liter of pooled material wasloaded into the system at a 500 ml volume and then concentrated to 250ml. Diafiltration was performed with 5 diavolumes (1250 ml) of ModifiedRinger's Solution+5% glycerol. Following diafiltration, the retentatewas concentrated to a final volume of 14 ml. Total process time wasapproximately 3.25 hours (not including sanitization time).Silver-stained SDS gels, slot blots, and infectivity assays confirmedthat the AAV preparation (which contained approximately 10¹⁰ replicativeunits) was substantially free of contaminating adenovirus as well asadenoviral and cellular proteins.

The following are results from such a procedure showing infectious andtotal virus titer as RU (replicative units) and DRP (DNAse-resistantparticles) respectively: 300K TFF Volume Total RU Total DRP P/I % RU %DRP Input Pool 1000 ml 8.9 × 10¹⁰ 3.1 × 10¹⁴ 3483 100 100 Purified Bulk12.5 ml 7.3 × 10¹⁰ 2.3 × 10¹⁴ 3103 82 74

The data presented in FIG. 12 illustrates the results of an AAVproduction run using tangential flow filtration after anion exchangecolumn. Material purified on the POROS 50 PI column was concentratedusing a 300,000 molecular weight cut-off membrane (Millipore PelliconXL). The concentrated material was diafiltered with five successivevolumes of Ringer's Balanced Salt Solution+5% glycerol. The material wasthen concentrated on the membrane 10-fold. FIG. 12, a half-tonereproduction of an SDS polyacrylamide gel stained with a silver stainsshows the highly-purified AAV capsid proteins, VP1(85 kD), VP2 (72 kD),and VP3 (62 kD), in the final purified bulk material.

As is apparent from the data presented herein, these second generationtechniques for the preparation and purification of AAV result insubstantially improved methods as compared with those describedpreviously.

Exemplary media for growing the Adenovirus helper and for preparing rAAVare detailed in the following Table: TABLE 2 Ad medium rAAV mediumINORGANIC SALTS CaCL 116.61 CuSO4*5H20 0.00125 0.00125 Fe(NO3)3*9H200.05 0.05 FeS04.7H20 0.417 0.417 KCL 311.8 311.8 MgCl2 28.61 MgSO4 48.84NaCl * * NaHC03 2200 2200 NaH2PO4.H20 62.5 62.5 Na2HP04 71.02 71.02Zn2SO4.7H20 0.4315 0.4315 OTHER COMPONENTS Glucose 4500 4500 HEPES 35753575 Hypozanthine Na 2.39 2.39 Linoleic acid 0.042 0.042 Lipoic acid0.105 0.105 Phenol Red, Na Salt Putrescine.2HCL 0.081 0.081 SodiumPyruvate 55 55 Pluronic Polyol F-68 100 100 AMINO ACIDS L-Alanine 4,4554,455 L-Arginine.HCL 273.9 273.9 L-Asparagine.H20 22.5 22.5 L-Aspartic19.95 19.95 L-Cysteine.HCL.H20 17.56 17.56 L-Cystine.2HCL 52.29 52.29L-Glutamic acid 22.05 22.05 L-Glutamine 657 657 Glycine 26.25 26.25L-Histidine.HCL.H20 73.48 73.48 L-Isoleucine 106.97 106.97 L-Leucine111.45 111.45 L-Lysine.HCL 163.75 163.75 L-Methionine 32.34 32.34L-Phenylalanine 68.48 68.48 L-Proline 17.25 17.25 L-Serine 36.75 36.75L-Threonine 101.05 101.05 L-Tryptophan 19.22 19.22 L-Tyrosine 91.7991.79 L-Valine 99.65 99.65 VITAMINS d-Blotin 0.00365 0.00365 D-CaPantothenate 2.24 1.00 Choline Chloride 8.98 8.98 Folic Acid 2.65 2.65myo-inositol 12.6 12.6 Niacinamide 2.0185 2.0185 Pyridoxal.HCL 2 2Pyridoxine.HCL 0.031 0.031 Riboflavin 0.219 0.219 Thiamine.HCL 2.17 2.17Thymidine 0.365 0.365 Vitamine B12 0.68 0.68* add appropriate amount of NaCl for osmolality of 300 mOsM (+ or −20mOsM)C. Purification of AAV Vector Using Heparin Sulfate Chromatography

As discussed above, chromatographic techniques can be employed tofurther purify and concentrate AAV preparations in acccordance with thepresent invention. By way of illustration, a preparation of AAV which isin crude form (e.g. lysate), or which has been eluted from ananion-exchange or cation-exchange column and/or concentrated bytangential flow filtration can be purified by binding to a columncomprising heparin sulfate. The AAV can then be eluted from such acolumn using a buffer containing a salt (e.g. a linear gradient ofNaCl).

As illustrative of the use of heparin sulfate chromatography, AAVobtained from a “PI” pool (as described below in Example 9) was firstconcentrated four-fold and diafiltered into TMEG+100 mM NaCl using a300K tangential flow filtration membrane. The concentrate was theninjected on a 1 ml heparin sulfate column (Pharmacia “Hi-Trap Heparin”column), and eluted using a linear gradient of NaCl.

FIG. 13 is a chromatogram showing the resulting concentration of AAV onthe heparin sulfate column. The sharp peak in absorbance at 280 nm(left-hand axis) at about 18 minutes elution time represents the AAVfraction as eluted from heparin sulfate with a linear gradient of 0 to1M NaCl (conductivity in ms shown on right-hand axis).

Example 8 Recombinant AAV Vector Production and Testing

In another set of production runs, we used 3-4×10⁹ cells grown in a CellFactory, using DMEM+10% FBS as the growth medium. Cells were infectedwith Ad at an MOI of about 20, and harvested at 72 hours post-infection.Harvested cells were suspended in TMEG+NaCl at a concentration of about5×10⁶ cells/ml. After mechanical lysis (microfluidization, 2 passes at8000 psi), lysates were treated with Benzonase (25 units/ml, 37 degreesC., one hour), and then filtered through a 5 micron filter (Pall ProfileII).

As an exemplary anion exchange column, we employed the POROS 50 PIcolumn (available from Perseptive Biosystems). Briefly, the filtrate wasloaded onto the column in about 100 ml and eluted with a gradient ofNaCl to 500 mM. Fractions determined (by infectivity assay) to containthe majority of the AAV were collected and pooled (referred to as the“PI pool”).

The PI pool was then diluted about 1:7 in TMEG and loaded on a 50 mlToso Haas SP650C column, and eluted with a gradient to 500 mM NaCl.Fractions determined (by infectivity assay) to contain the majority ofthe AAV were collected and pooled (referred to as the “SP pool”). The SPpool was concentrated using a Centriprep 10K filter, and then wassterilized by passage through a 0.2 micron filter.

The results revealed that the recombinant AAV was essentially free ofdetectable infectious adenovirus (as determined by limit of detectionanalysis with serial amplification on 293 cells and TCID50 assay). Thepreparation was also essentially free of adenoviral DNA (as determinedby slot blot analysis), essentially free of cellular proteins (asdetermined by SDS-PAGE gel analysis), of cellular DNA (determined by PCRanalysis), and was also essentially free of phenotypically wild-type AAV(as determined by serial amplification and Southern analysis).

Example 9 The Enhancement of AAV Production by Nutritional Stress

As discussed above, it is believed that AAV production can be enhancedusing any of a variety of agents and/or conditions that effectivelystress (or de-optimize) growth or metabolism of the AAV producer cells.In this example, it is shown that the depletion of certain amino acidsas occurs during culture is associated with a relative enhancement inAAV production; and, conversely, that media supplements to remove thenutritional stress actually result in a dramatic reduction in vectoryield.

(a) Nutritional Stress During Batch and Perfusion Culture

JL14 cells were inoculated at about 4×10⁵ cells/ml in 2 literbioreactors and grown in the rAAV medium shown in Table 2 in eitherbatch mode or by perfusion (using tangential flow filtration, day 1 at0.4 volumes/day, days 2-3 at 1.2 vol./day, day 4 at 2 vol./day and day 5at 4 vol./day). Cultures were monitored for cell density, glucose,lactate and amino acids using standard techniques.

The analyses revealed that cell density peaked in batch culture at 1×10⁶cells/ml on day 2, and in perfusion culture at 8×10⁶ cells/ml on day 6.Glucose was not limiting in either case (>1 g/l) and lacate was notinhibitory.

However, amino acid analysis revealed that both glutamate and aspartatewere rapidly depleted in both batch and perfusion cultures, as shown inthe following Tables: TABLE 3 Amino acid analysis of BATCH culturemedium (time course - μmol/L MW day 0 day 1 day 2 day 3 day 4 AsparticAcid 133 96 10 4 9 7 Threonine 119 687 644 606 552 533 Serine 105 271230 157 117 98 Asparagine 132 130 113 96 68 69 Glutamic 147 90 2 1 1 1Acid Glutamine 146 3424 2987 2450 1989 1843 Proline 115 135 143 162 164185 Glycine 75 288 241 194 151 130 Alanine 89 189 306 438 644 681 Valine117 692 631 518 417 342 Cystine 121 143 133 120 107 99 Methionine 149160 132 100 74 58 Isoleucine 131 617 531 383 264 182 Leucine 131 645 538374 248 161 Tryosine 181 407 379 355 329 315 Phenylalanine 165 323 289259 231 214 Tryptophan 204 47 41 33 28 26 Ammonia 17 760 816 941 10211033 Ornthinine 71 89 110 128 144 Lysine HCl 572 521 463 415 384Histidine 155 276 257 243 229 211 Arginine 174 1020 943 870 791 747

TABLE 4 Amino acid analysis of PERFUSION culture medium (Time course -μmol/L) MW day 0 day 1 day 2 day 3 day 4 day 5 day 6 Aspartic Acid 13395 12 5 10 10 10 10 Threonine 119 709 691 560 596 651 641 657 Serine 105281 264 147 156 180 199 185 Asparagine 132 130 124 75 78 109 119 119GlutamicAcid 147 88 1 0 1 1 1 0 Glutamine 146 3525 3299 2517 2640 29062986 3082 Proline 115 145 165 163 174 177 157 171 Glycine 75 304 267 189205 217 227 230 Alanine 89 190 340 341 384 423 333 330 Valine 117 678635 485 500 532 551 561 Cystine 121 141 136 112 118 123 119 118Methionine 149 157 133 91 99 107 108 107 Isoleucine 131 616 543 369 401430 432 442 Leucine 131 649 554 364 400 430 438 444 Tryosine 181 413 398328 355 379 373 386 Phenylalanine 165 336 316 244 268 291 287 292Tryptophan 204 58 47 36 41 48 44 47 Ammonia 17 831 1182 956 1202 1219931 990 Ornthinine 42 97 74 115 112 56 44 Lysine HCl 718 651 528 594 643617 628 Histidine 155 284 310 223 243 262 266 265 Arginine 174 1058 948826 901 978 974 1016

(b) Nutritional Stress Associated With Enhanced AAV Production

Follow-up studies were performed to confirm the importance of therelative paucity of glutamate and aspartate in the culture media. JL14cells were taken from a spinner flask and divided into two sets. Eachset was innoculated with 3×10⁹ infectious units of 170-37 Ad 5. One setof cells was resuspended at 10⁶ cells/mL in rAAV medium (Table 2)containing 10% FBS and 1% L-glutamine (300 mL). The other wasresuspended in rAAV medium containing 10% FBS, 1% L-glutamine, 10 mg/Laspartatic acid, and 110 mg/L glutamic acid.

Each set was incubated at 37 degrees for 72 hours in a spinner flask.The cells were harvested, microflidized twice at 8000 psi, Benzonased,plated into an infectivity assay, harvested and probed.

Results showed that the control spinner flask produced 6.2 RUs per cell.The spinner flask supplemented with aspartic and glutamic acid produced0.94 RUs per cell.

This indicates that when depletion of aspartic acid and glutamic acid isprevented by providing these amino acids in excess, rAAV production iscompromised due to the failure to subject the cells to nutritionalstress.

Further tests were performed using a HeLa-derived cell line D6 which hasan integrated rAAV vector (ITR-(CMV promoter)-(β-gal reportergene)-ITR), as well as copies of the wild-type AAV rep and cap genes.

The cells were seeded at 5×10⁶ cells per T-225 flask in 30 mL completeDMEM (10% FBS, 2 mM L-Glutamine), and incubated at 37 degrees in 10% CO₂for 2 days, whereupon the cells reached a density of 2×10⁷ cells perflask. Cells in two duplicate flasks were infected with Ad 5 at an MOIof 10. One flask contained complete DMEM, the other contained completeDMEM supplemented with 5×aspartic acid and glutamic acid. Cells wereharvested and counted after 72 hours of culture.

The complete DMEM yielded 2.6×10⁷ cells with 88% viability. Theaspartate/glutamate supplemented medium yielded 3.8×10⁷ cells with 91%viability. Cells were resuspended, sonicated, treated with Benzonase (25U/ML), clarified, and assayed by slot blot analysis.

Results were as follows: D6 virus was produced in complete(unsupplemented) DMEM at 1.8×10¹⁰ DRP/mL (1800 DRP per cell). D6 viruswas produced in aspartate/glutatmate supplemented DMEM at 1.4×10⁹ DRP/mL(140 DRP/cell).

Example 10 Recombinant AAV Vector Production Under Serum Stress

As an example of rAAV production under stress conditions, we have usedreduced-serum stress in conjunction with techniques as described above.Briefly, JL-14 cells were grown in spinner flasks in modified DMEM +10%FBS in continuous serial culture mode, and were split every 3-4 days.Cells from suspension culture were placed into 16 Nunc Cell Factories,10-stack, at 3×10⁸ cells/factory on a three- to four-day rotation. Themedium used for growth had a ten-fold reduction in serum (i.e. DMEM+1%FBS) thereby placing the cells under serum stress.

At 24 hours after seeding, the medium in the factories was removed andfresh medium containing 3×10⁹ Ad units/ml was added. After 72 hours ofculture at 37 degrees, the cells were dislodged from the factories bygentle tapping, medium containing cells was collected and the cells werepelleted and resuspended in TMEG+100 mM NaCl, and then lysed by passagethrough a microfluidizer at 8000 psi. The lysate was clarified through a5 micron filter and the clarified lysate was loaded on a 500 ml PI anionexhange column. The column was eluted with a gradient of increasing NaCl(up to 500 mM) in TMEG buffer. Fractions were collected and assayedusing a Clone 37 assay as described by Allen et al. (WO96/17947, supra).The fractions containing most of the AAV vector were then pooled andconcentrated 10-fold using a Centriprep centrifugal concentrator at1000×g for 30 minutes. The concentrated material was dialyzed againstRinger's Balanced Salt Solution with 5% glycerol, and stored at −70degrees C. The AAV was assayed by the Clone 37 assay, as well as by slotblot and SDS-PAGE. The material may also be assayed for the presence ofadenovirus, adenoviral proteins, and cellular DNA, as well as otherpotential contaminants.

FIG. 11 shows the results obtained using GAK-0003 producer cells set upin T-225 flasks at 10⁷ cells per flask, and innoculated on Day 2 withDAB-003 adenovirus at an MOI of 10. Different flasks were cultured for72 hours at 37 degrees in fresh DMEM containing a different percentageof FBS, as shown in the figure. On Day 5, each flask was harvested, thecells were counted, resuspended, sonicated, Benzonased, and plated tomeasure vector production as before.

Optimal vector producton was observed at a FBS percentage of 1%.Accordingly, medium that is deficient in FBS (less than 2.5%, preferablyless than 2% but more than 0%) is preferred as a condition forsubjecting the producer cells to serum stress.

Example 11 Recombinant AAV Vector Production Under pH Stress

As a further example of rAAV production under stress conditions, we haveused pH stress in conjunction with techniques as described above.Briefly, AAV producer cells were grown in bioreactors as describedabove. Cells were then infected with Ad5 at MOI=10 and inoculated intolow-serum media (as in Example 11) in suspension in 1.5 literbioreactors. Cultures were maintained at various elevated pH levels(from 7.2 to 8.0). Cultures were then monitored daily for cell number,viability, glucose consumption, lactate production, pH, osmolarity andAAV production. As shown below, there was an increase in AAV productionwhen the pH was elevated to 7.4; coupled with an even more dramaticincrease in the number of AAV particles released into the supernatant(which increased as pH was elevated): Cell- % in Culture associatedSupernatant Total % Cell- Super- pH Particles Particles Particlesassociated natant 7.2 4.70E + 12 1.90E + 09 4.70E + 12 100% 0% 7.46.50E + 12 1.30E + 13 1.95E + 13 33% 67% 7.6 3.40E + 12 1.50E + 131.84E + 13 18% 82% 8.0 1.30E + 12 1.50E + 13 1.63E + 13 8% 92%

In sum, as pH was raised, we observed a sharp increase in the number ofAAV particles released into the supernatant, and a shift in thepercentage of supernatant:cell-associated particles (from nearly allcell-associated at pH 7.2 to mostly supernatant (92%) at pH 8.0). Theability to recover AAV particles directly from the supernatant withoutthe need for lysing the producer cells represents a powerful advantagein terms of AAV production and purification. AAV isolated from thesupernatant using pH stress can be readily concentrated and purifiedusing techniques as described herein (e.g. ion-exchange chromatographyand/or tangential-flow filtration).

Example 12 General Methods for Additional rAAV Release Experiments

Quantitation of rAAV Titers in Vector Preparations: Slot Blot Assay

The rAAV DNA slot blot assay was conducted as follows. Aliquots ofsamples were digested with nuclease to remove unencapsidated DNA. Thesamples were then denatured in 0.4M NaOH, 10 mM EDTA with 1.0 μg/mlsalmon sperm DNA at 65° C. Samples and rAAV standards were diluted andfiltered onto nylon membranes using a slot blot manifold and washed with0.4M NaOH. The filter was hybridized with a ³²P-labeled human CFTR cDNArestriction fragment. This probe detects an approximately 1.5 kbfragment from the AAVCF vector (corresponding to the predicted 1.488 kbEcoRI fragment).

Microtiter Infectivity Assay to Measure rAAV

The microtiter infectivity assay was conducted as previously described.Atkinson et al. (1998) Nucleic Acids Research 26(11): 2821-2823.Briefly, a high-throughput microtiter infectivity assay to measureinfectious virus was conducted as follows. Aliquots (10 μl) of seriallydiluted cell-free supernatants were inoculated onto HeLa clone 37 cellsgrown in 96-well microtiter plates. After three days, infected cellswere treated and lysed with a denaturation solution (addition of1/10^(th) volume of 4.0 M NaOH, 10 μg/ml salmon sperm DNA and 100 mMEDTA). Lysate was transferred to a Silent Monitor BiodyneB plate (Pall)and vacuum filtered onto the nylon membrane. The membrane was washed,denatured, hybridized with a ³²P-labeled human CFTR cDNA restrictionfragment. This probe detects an approximately 1.5 kb fragment from theAAVCF vector (corresponding to the predicted 1.488 kb EcoRI fragment).Vector replication was quantitated relative to an endogenous genomicCFTR band and is expressed as replication units. One replication unit(RU) is defined as a signal intensity equivalent to that of theendogenous genomic CFTR band which is approximately 1.8 kb. Linearregression of serially diluted known vector standards was used toextrapolate and calculate vector concentration in samples.

Production Media

Tables 5 and 6 provide concentrations of components (in mg/l) of mediasuitable for growing cells and producing rAAV (blanks indicate zeroconcentration). Generally, it is preferable to have reduced serumlevels. For example, the media used in these experiments contained about1% fetal bovine serum (FBS). TABLE 5 Concentration INORGANIC SALTS CaCl2anhydrous 200 Fe(NO3)3*9H20 0.1 KCL 400 MgSO4*7H20 200 NaCl 4675 NaHC031200 NaH2PO4.H20 125 OTHER COMPONENTS Glucose 8500 HEPES 3575 PhenolRed, Na Salt sodium pyruvate 110 calcium pantothenate 6 choline chloride6 folic acid 6 inositol 11 nicotinamide 6 pyridoxal HCl 2 pyridoxine HCl4 riboflavin 0.6 thiamine HCl 6 F-68 500 AMINO ACIDS L-Alanine 8.9L-Arginine.HCL 236.9 L-Asparagine.H20 17 L-Aspartic acid 13.3 L-Cystine72 L-Glutamic acid 14.7 L-Glutamine 1168 Glycine 37.5L-Histidine.HCL.H20 84 L-Isoleucine 157.3 L-Leucine 157.2 L-Lysine.HCL218.7 L-Methionine 45.1 L-Phenylalanine 99 L-Proline 11.5 L-Serine 52.5L-Threonine 142.8 L-Tryptophan 26.2 L-Tyrosine 108 L-Valine 140.4

TABLE 6 Component Concentration, mg./L CaCl2 200 Fe(NO3)3.9H2O 0.1 KCl400 MgSO4.7H2O 200 NaCl 4675 NaHCO3 1200 NaH2PO4.H2O 125 glucose 4500HEPES 3575 sodium pyruvate 110 calcium pantothenate 4 choline chloride 4folic acid 4 inositol 7 nicotinamide 4 pyridoxal HCl pyridoxine HCl 4riboflavin 0.4 thiamine HCl 4 F68 500 L-alanine L-arginine HCl 84L-asparagine L-aspartic acid L cysteine 48 Lglutamic Lglutamine 876glycine 30 I-histidine-HCl.H2O 42 Lisoleucine 104.8 L-Leucine 104.8L-Lysine HCl 146.2 L-methionine 30 L-phenylalanine 66 L-proline L-serine42 L-threonine 95.2 L-tryptophan 16 Ltyrosine 72 Lvaline 93.6

Example 13 Effect of PH on rAAV Vector Particle Production in ProducerCell Lysates

JL14 cells were inoculated at about 3×10⁵ cells/ml in a 3 literbioreactor and grown in 1.5 liters of the rAAV medium shown in Table 5.Two days after inoculating the culture medium in the bioreactor withJL14 cells, the bioreactor was perfused with fresh medium, beginning at0.4 volumes per day, then doubling this amount every 24 hoursthereafter. After 5 days, or when the cell density reached 6×10⁶cells/ml, 3×10⁸ cells were removed and grown under standard conditions,i.e, allowing the pH to vary. The cells remaining in the Bioreactor wereconcentrated in a volume of 750 ml culture medium, and a three volumemedium exchange was performed with production medium (i.e., medium as inTable 2) at pH 7.2 to exchange the medium, so that the final volume ofcell culture was 750 ml. Adenovirus type 5 was grown from a stockobtained from the American Type Culture Collection (Manassas, Va.).Adenovirus was diluted into 750 ml production medium and added to thecells (multiplicity of infection (MOI)=10), bringing the final volume to1.5 liters. Infection with adenovirus was allowed to proceed for onehour at 37° C.

After allowing infection to proceed, 1×10⁵ cells were transferred toeach of 5 separate spinner flasks. The volume in the five flasks wasbrought up to 1.5 liters with production medium at pH 6.6, 7.0, 7.2,7.4, and 7.8, respectively. The contents of the spinner flasks weretransferred to separate bioreactors, which were then maintainedindividually at pH 6.6, 7.0, 7.2, 7.4, and 7.8. Other culture mediumparameters were as follows: temperature=37° C.; dissolved oxygenconcentration (DO₂)=30%; and agitation=150 rpm. Temperature, pH, DO₂,cell density, osmolarity and glucose/lactate were monitored daily. Cellsamples were harvested on day 2 and day 3 post-infection and lysed. Thecell lysates were assayed by DNAse-resistant particles (DRP) slot blotassay and by the microtiter infectivity assay.

FIGS. 14A and 14B are bar graphs depicting the results of two separateexperiments, expressed as DRP per cell at the various pH levels. Solidbars represent DRP/cell at day 2 post-infection; hatched bars representthe DRP/cell at day 3. The DRP/cell in the cultures maintained at pH7.2, pH 7.4, and pH 7.8 decreased dramatically from day 2 to day 3post-infection, while this reduction was not as pronounced in thecontrol cell culture. The total cell density did not change appreciablyunder these culture conditions.

Example 14 Effect of PH on Release of rAAV Vector Particles into theCell Culture Medium

To further investigate the effects of pH on rAAV vector production, JL14cells were grown in a perfusion bioreactor as described above (usingmedia described in Table 5) to a density of 10⁷ cells/ml. The cellculture was concentrated to a volume of 750 ml and the medium exchangedby performing 3 diavolumes. The total volume was brought to 1.5 literswith production medium containing adenovirus at a MOI of 10. Infectionwas allowed to proceed for one hour.

After allowing infection to proceed, 1×10⁵ cells were transferred toeach of 5 separate spinner flasks. The volume in the five flasks wasbrought up to 1.5 liters with production medium at pH 7.2, 7.4, 7.6,7.8, 8.0, and control flasks (pH not maintained at the starting level).The contents of the spinner flasks were transferred to separatebioreactors, which were then maintained individually at pH 7.2, 7.4,7.6, 7.8, 8.0. Bioreactors maintained the pH at the stated level ±0.05pH units. Other culture medium parameters were as follows:temperature=37° C.; dissolved oxygen concentration (DO₂)=30%; andagitation=150 rpm. Cells and culture supernatants (culture media) wereharvested on days 2 and 3.

The results are shown in FIGS. 15A and 15B. FIGS. 15A and 15B are bargraphs and depict the results, expressed as total DRPs, of rAAVproduction in bioreactors maintained at various pH levels. Percentagesabove each bar are percentages of total DRPs in the cell lysate. Thesolid portion of each bar represents DRPs in cell lysates, while thehatched portion of each bar represents the DRPs in the cell culturemedium. On day 2 post-infection, 29% of the total DRPs were in theculture medium of the culture maintained at pH 8.0, while on day 3,post-infection, the percent of total DRPs in the culture medium rose to92%. On day 3 post-infection, the percentage of total DRPs in theculture medium was 67% at pH 7.4, 82% at pH 7.6, 73% at pH 7.8 and 92%at pH 8.0. Cultures maintained at pH 7.2 did not yield any DRPs in thecell culture medium in this experiment.

The day 2 and day 3 post-infection cell lysates and culture media fromthe bioreactors maintained at pH 7.2, 7.4, 7.6, 7.8, and 8.0 wereassayed for replication units (RU) using an infectivity assay. The dataare shown in FIGS. 16A and 16B. The total cell density did not changeappreciably under these culture conditions.

FIGS. 16A and 16B are bar graphs depicting the total replication units(RU) assayed at day 2 (FIG. 16A) and day 3 (FIG. 16B) post-infection inthe culture media-(hatched portion of each bar) and cell lysates (solidportion of each bar) when cultures were maintained at the indicated pHlevels. Percentages above each bar indicate the percentage of total RUsin the cell lysate. These data demonstrate that the rAAV particlesreleased into the cell culture medium are functional in an infectivityassay.

FIG. 17 is a bar graph depicting the particle:infectivity (P/I) ratio ofrAAV particles harvested from cell lysates (solid portion of each bar)and cell culture medium (hatched portion of each bar) at day 3post-infection from bioreactors maintained at the indicated pH levels.These date indicate that the majority of the rAAV vector released intothe cell culture medium is infectious.

Example 15 Effect of Osmolality on Release of rAAV Vector Particles intothe Cell Culture Medium

To assess the effects on release of rAAV into the cell culture medium ofstarting osmolality of the culture medium, JL14 cells were grown inbioreactors and infected with adenovirus essentially as described inExample 14. The starting osmolality in the individual bioreactors was130, 200, 300, 400, and 500 mOsm (using NaCl), respectively. In eachreactor, the pH was maintained at pH 8.0 (±0.05); temperature=37° C.;DO₂=30%; and agitation=150 rpm. On days 2, 3, and 4 post-infection, celllysates and cell culture media were collected and analyzed for rAAVvector production.

The results are shown in FIGS. 18-20.

FIGS. 18A, 18B, and 18C are bar graphs depicting the total DRPs in celllysates (solid portion of each bar) and cell culture media (hatchedportion of each bar) on day 2 (FIG. 18A), day 3 (FIG. 18B), and day 4(FIG. 18C) post-infection in bioreactors in which the cell culture mediacontained the indicated starting osmolality. Percentages above each barindicate the percentage of total DRPs in the cell lysate. These datashow that when the cell culture medium has a starting osmolality of 300mOsm, 41%, 59%, and 80% of the total DRPs are in the cell culture mediumat day 2, 3, and 4, respectively. A starting cell culture mediumosmolality of 300 mOsm gave the maximum percentage of total rAAV vectorin the cell culture medium, compared with other starting osmolalitiestested. The total cell density did not change appreciably under theseculture conditions.

FIGS. 19A, 19B, and 19C are bar graphs depicting the total RUs in celllysates (solid portion of each bar) and cell culture media (hatchedportion of each bar) on day 2 (FIG. 19A), day 3 (FIG. 19B), and day 4(FIG. 19C) post-infection in bioreactors in which the cell culture mediacontained the indicated starting osmolality. Percentages above each barindicate the percentage of total RUs in the cell lysates. These dataindicate that the rAAV vector released into the medium is infectious.

FIG. 20 is a bar graph depicting the P/I ratio of rAAV particles in cellculture media at days 3 and 4 from bioreactor cultures with theindicated starting osmolalities.

Example 16 Effect of Temperature on Release of rAAV Vector Particlesinto the Cell Culture Medium

JL14 cells were grown in bioreactors and infected with adenovirusessentially as described in Example 2. Cells were transferred tobioreactors maintained individually at 31° C., 34° C., 37° C., 39° C.,and 42° C., respectively. These temperatures were maintained ±0.5° C.The pH in each reactor was maintained at 8.0 (±0.05); agitation=150 rpm;DO2=30%. On days 2, 3, and 4 post-infection, cell lysates and cellculture media was analyzed for rAAV particles.

The results are shown in FIGS. 21 and 22.

FIGS. 21A-C are bar graphs depicting the total DRPs in cell lysates(solid portion of each bar) and cell culture media (hatched portion ofeach bar) on day 2 (FIG. 21A), day 3 (FIG. 21B), and day 4 (FIG. 21C)post-infection in bioreactors in which the cell culture media wasmaintained at the indicated temperature. Percentages above each barindicate the percentage of total DRPs in the cell lysate. The data showthat when the culture medium was maintained at 39° C., 66%, 67%, and 57%of the total DRPs were found in the cell culture medium on days 2, 3,and 4, respectively. The data further indicate that a higher percentageof DRPs was found in the cell culture medium when the culture media wasmaintained at 39° C., compared to 37° C. or 42° C.

FIGS. 22A-C are bar graphs depicting the total RUs in cell lysates(solid portion of each bar) and cell culture media (hatched portion ofeach bar) on day 2 (FIG. 22A), day 3 (FIG. 22B), and day 4 (FIG. 22C)post-infection in bioreactors in which the cell culture media wasmaintained at the indicated temperature. Percentages above each bar inFIG. 22A indicate the percentage of total RUs in the cell culturemedium. These data show that when the culture medium was maintained at39° C., 80%, 97%, and 98% of total RUs were found in the cell culturemedium on days 2, 3, and 4, respectively. The total cell density did notchange appreciably under these culture conditions.

Example 17 Effect of Culture Medium Supplements on Release of rAAVVector Particles into the Cell Culture Medium

JL14 cells were grown in bioreactors and infected with adenovirusessentially as described in Example 2. Cells were transferred tobioreactors containing various media, as follows: (1) DMEM; (2) DMEM+4g/liter glucose; (3) DMEM+4 g/liter glucose+4 mM glutamine; (4) DMEM+4g/liter glucose+4 mM glutamine+amino acids+vitamins (“complete”); (5)2×DMEM. All starting osmolalities were adjusted to 285-300 mOsm. Otherparameters were as follows: temperature maintained at 39° C.; pHmaintained at 8.0; DO₂=30%; and agitation=150 rpm. Three dayspost-infection, the cell culture supernatant was assayed for rAAV vectorparticles.

The results are shown in FIGS. 23, 24, and 25.

FIG. 23 is a bar graph depicting the total DRPs in the culture mediathree days post-infection in cultures grown in the various mediaindicated.

FIG. 24 is a bar graph depicting the RUs in the culture media three dayspost-infection in cultures grown in the various media indicated.

FIG. 25 is a bar graph depicting the P/I ratio of viral particles in thecell culture media when cultures were grown in the various mediaindicated.

Example 18 Effect of Osmolality and Conductivity on Release of rAAVVector Particles into the Cell Culture Medium from Attached CellCultures Example 18A Vector Release from Attached Cell Cultures

Media. The media in Table 7 was used in the experiments of this Example.TABLE 7 Base Media Formulation DMEM liquid DMEM Powder (BioWhittaker)(BioWhittaker) Component mg/L mg/L CaCl2(anhydrous) 200 CaCl2.2H2O264.86 Fe(NO3)3.9H2O 0.10 0.10 KCl 400 400 MgSO4(anhydrous) 97.6MgSO4.7H2O 200 NaCl 6400 6400 NaHCO3 3700 NaH2PO4 108.69 NaH2PO4.H2O 125Glucose 4500 4500 Phenol Red 15.00 Phenol Red.Na 15.34 Sodium Pyruvate110 L-Arginine. HCl 84.00 84.00 L-Cysteine 48.00 L-Cysteine. 2HCl 62.58L-Glutamine 584.00 584.00 Glycine 30.00 30.00 L-Histidine.HCl.H2O 42.0042.00 L-Isoleucine 104.80 104.80 L-Leucine 104.80 104.80 L-Lysine. HCl146.20 146.20 L-Methionine 30.00 30.00 L-Phenylalanine 66.00 66.00L-Serine 42.00 42.00 L-Threonine 95.20 95.20 L-Tryptophan 16.00 16.00L-Tyrosine 72.00 L-Tyrosine.2Na 103.79 L-Valine 93.60 93.60D-Ca-Pantothenate 4.00 4.00 Choline Chloride 4.00 4.00 Folic Acid 4.004.00 i-Inisitol 7.00 7.00 Nicotinamide 4.00 4.00 Pyridoxine.HCl 4.004.00 Riboflavin 0.40 0.40 Thiamine.HCl 4.00 4.00

To assess the effects of changes in osmolality and conductivity onrelease of rAAV vector particles into the cell culture medium ofattached cell cultures, JL 14 cells were innoculated at about 1×10⁷cells per flask in T-225 cells and grown in 54 ml of the medium shown inTable 7 with additional formulation as described below in Table 8 (FBSand L-glutamine were supplemented to 1%) overnight at 37° C., 10% CO₂,pH 7.2. Twenty four hours post seeding of the cells in the T225 flasksthe media was removed and replaced with 54 ml of each test mediadescribed below and infected with Ad5 at an MOI of 10. T225 cultures asdescribed were maintained for 72 hours at 37° C., 10% CO₂ to allow forrAAV production. Three days later cells and supernatant were collectedfrom each test flask and total cell density as well slot blot andinfectivity assays comparing released rAAV (supernatant) and cellassociated rAAV (lysed) were performed. “Adjusted osmolality” refers tothe osmolality after adding indicated solute, which represents theosmolality at the beginning of the experiment (i.e., upon infection withadenovirus). TABLE 8 Media Formulation Table Solute Initial Added mOsmAdjusted Conductivity Media Base mOsm Solute Added mOsm mS Liquid DMEM359 None 0 359 13.30 Powdered DMEM 363 None 0 363 13.75 Powdered DMEM363 NaCl 27 390 14.88 Powdered DMEM 363 NaCl 66 429 16.55 Powdered DMEM363 Sorbitol 28 391 13.46 Powdered DMEM 363 Sorbitol 63 426 13.33

The results are shown in FIGS. 27-30. The total cell density is notappreciably different if the media is formulated with NaCl or sorbitol(FIG. 27). The maximum percentage of total rAAV vector was released intothe culture media (in terms of DRP) when the adjusted osmolality of themedia is formulated at approximately 429 mOsm using NaCl (resulting in aconductivity of 16.55 mS) compared with the other starting osmolalitiesand conductivities tested (FIG. 28). In contrast, there were no releasedrAAV vector particles detected when sorbitol with adjusted osmolality of426 (conductivity of 13.33 mS) (FIG. 28).

Similarly, when vector production was measured in terms of RUs, themaximum percentage of infectious rAAV vector was released in the culturemedium when the starting osmolality of the media is formulatedapproximately 429 mOsm with NaCl resulting in a conductivity of 16.55 mS(about 80%) compared with the other starting osmolalities andconductivities tested (FIG. 29). In contrast, less than 20% of total RUswere in the culture supernatant when sorbitol was used to adjust theosmolality to 426 mOsm (conductivity of 13.33 mS).

P/I (particle to infectivity ratio) data indicate that the majority ofthe rAAV vector released into the cell culture medium is infectious(FIG. 30).

Example 18B Effect of Varying Times of Adjusting Osmolality andConductivity on Vector Release from Attached Cell Cultures

JL 14 cells were inoculated at about 1×10⁷ cells per flask in T-225cells and grown in 54 ml of the medium shown in Table 7 overnight at 37°C., 10% CO₂, pH 7.2. Twenty four hours post seeding of the cells in theT225 flasks the media was removed and replaced with 54 ml media listedin Table 9 and infected with Ad5 at an MOI of 10. At various time pointsthe media was adjusted with 5M NaCl to achieve a final osmolarity of 450mOsm. T225 cultures as described were maintained for 72 hours at 37° C.,10% CO₂ to allow for rAAV production. Three days later cells andsupernatant were collected from each test flask and total cell densityas well slot blot infectivity assays comparing released rAAV(supernatant) and cell associated rAAV (lysed) were performed. TABLE 9Media Formulations Solute Day 3 Starting Added Day Solute mOsm FinalConductivity Media Base mOsm Solute Adjusted Added mOsm mS Powdered DMEM363 None ND 0 363 12.6 Powdered DMEM 363 NaCl 0 87 450 17 Powdered DMEM363 NaCl 1 87 450 16.9 Powdered DMEM 363 NaCl 2 87 450 16.9 PowderedDMEM 363 NaCl Day 3 (−4 hr) 87 450 16.9 Powdered DMEM 363 NaCl Day 3 (−1hr) 87 450 16.8

The results are shown in FIGS. 31-32. As shown in FIG. 31A, the majorityof DRPs (70%) were released into the media with adjustment of theosmolality and corresponding conductivity at day 2 of rAAV productionand that adjustment of the osmolality to 450 mOsm and conductivity withNaCl 4 hours prior to harvest on day 3 results in a majority of DRPsreleased in the cell culture medium (56%). A significant increase inrelease of rAAV vector was observed for all conditions in whichosmolality (and thus conductivity) was adjusted after day 0 as comparedto control.

The data demonstrate the majority of infectious rAAV vector (RUs) werereleased into the media with adjustment of the osmolality andcorresponding conductivity at any time during rAAV production (FIG.31B). Only 13% of total RUs were released in the control 363 mOsmculture, while the 450 mOsm cultures released 47%, 69%, and 74% for day0, 1, and 2 respectively. As little as 1-4 hours prior to harvest on day3 adjustment of the osmolality and conductivity to 450 mOsm with NaClresults in a majority of infectious RUs released in the cell culturemedium (FIG. 31B).

P/I (particle to infectivity ratio) data indicate that the majority ofthe rAAV vector released into the cell culture medium is infectious(FIG. 32).

Example 19 Effect of Osmolality and Conductivity on Release of rAAVVector Particles into the Cell Culture Medium from Suspension CellCultures

To further assess the effects of starting osmolality and conductivity ofthe culture cell medium on the release of rAAV into the cell culturemedium from suspension cell cultures, JL14 cells were grown inbioreactors and infected with adenovirus essentially as described inExample 14 using media as described in Table 5. The adjusted osmolalityin the individual bioreactors (at the beginning of the experiment) wasas described in Table 10. In each reactor, the pH was maintained at pH8.0 (0.05); 37° C.; DO2=30%; and agitation 150 rpm. The pH of 8.0 wasmaintained by the controlled addition of sodium carbonate to the culturemedium. On days 1,2, and 3 cultures were analyzed for conductivity,osmolality, total cell density, and glucose consumption. On days 2 and 3post infection, cell lysates and cell culture media were collected andanalyzed for rAAV vector production. TABLE 10 Media formulations forsuspension cell culture. Starting Added Solute mOsm Adjusted mOsm SoluteAdded mOsm 180 NaCl 70 250 180 NaCl 120 300 180 NaCl 170 350 180Sorbitol 20 200 180 Sorbitol 120 300 180 Sorbitol 170 350

The results are shown in Tables 11 and 12 and FIGS. 33 and 34.

Table 11 summarizes the change in osmolality of the bioreactor culturesduring rAAV vector production due to the controlled addition of sodiumbicarbonate to the culture to control the pH of the culture to pH 8.0(0.05). The data indicate that the changes in osmolality over time werenot appreciably different between the NaCl formulated and sorbitolformulated cultures. Generally, osmolality increased over time in allcultures. TABLE 11 Change in osmolality of the culture over time InitialAdded Culture Day 0 Day 1 Day 2 Day 3 Solute mOsm mOsm mOsm mOsm mOsmNaCl 250 265 307 367 396 NaCl 300 302 375 464 460 NaCl 350 348 402 454485 Sorbitol 200 200 255 313 350 Sorbitol 300 302 375 464 460 Sorbitol350 336 406 489 530

Table 12 depicts the change in conductivity of the bioreactor culturesduring rAAV vector production due to the controlled addition of sodiumbicarbonate to the culture to control the pH of the culture to pH 8.0(0.05). The data shows the greatest increase in conductivity of the NaClformulated bioreactor cultures over time compared to sorbitol formulatedcultures which did not change appreciably over time. TABLE 12 Change inconductivity of the culture over time Initial Added Culture Day 0 Day 2Day 3 Solute mS mS Day 1 mS mS mS NaCl 250 8.48 10 12.25 13.29 NaCl 30010.15 12.64 14.39 15.52 NaCl 350 12.38 14.12 16.26 17.28 Sorbitol 2005.3 7.54 9.35 10.51 Sorbitol 300 5.09 7.11 9.05 10.29 Sorbitol 350 4.887.48 10.04 11.5

The total cell density remained generally constant over the cultureperiod whether the cultures were formulated with NaCl or sorbitol(Compare FIGS. 33A and 33B). The metabolic rate of the cultures asmeasured by glucose consumption is equivalent between the NaCl andsorbitol formulated cultures is also equivalent (compare FIGS. 34A and34B).

The data evaluating the amount of vector released into the media (byDRPs) show that more DRPs are released into the media when NaCl is usedto formulate the media rather than sorbitol (e.g., 27% vs. 4% on day 2;48% vs. 23% at day 3) (FIGS. 35A and 35B). In terms of RUs,NaCl-formulated cultures released more rAAV at both day 2 and 3 (FIGS.35C and 35D).

Amount of rAAV released as indicated by RUs shows that more rAAV vectorper cell is released into the medium in the cultures formulated withNaCl as opposed to sorbitol as well as the majority of infectious rAAVvector (FIG. 36). 98% or 105.8 RUs were produced per cell in cultureswith a starting formulation of 300 mOsm NaCl at day 3 compared to 43% or3.6 RUs per cell for 350 mOsm starting sorbitol formulated cultures(FIG. 36).

P/I data indicate that the majority of the rAAV vector released into thecell culture medium is infectious (FIG. 37).

As indicated in FIGS. 33 and 34, the increase in rAAV vector releasedinto culture medium for NaCl formulated cultures as opposed to sorbitolformulated cultures was not due to a significant difference in metabolicrate (glucose consumption), total cell density, or osmolality of thecultures as there were no appreciable differences in the cultures.However, the cultures did differ in their conductivity (Table 12).Compare, for example, the NaCl formulation at a starting osmolality of250 mOsm with a conductivity of 10.00 mS that increased to 13.29 mS byday 3 to the sorbitol 300 mOsm starting formulation with a conductivityof 7.11 mS that increased to 10.29 mS by day 3. Generally, the NaClformulations demonstrating a range of conductivities betweenapproximately 10 and 15 mS demonstrate the greatest percentage releaseof rAAV vector into the supernatant.

Table 13 is an example of cell culture medium suitable for propagating,maintaining and/or expanding producer cells prior to infection withhelper virus (i.e., suitable for maintaining seed train for production).TABLE 13 Culture Medium, 1 Liter Bottle Component mg/L CuSO₄ * 5H₂O0.0013 Fe(NO₃)₃ * 9H₂O 0.05 FeSO₄ * 7H₂O 0.42 KCI 311.8 NaHCO₃ 2200Na₂HPO₄ * 7H₂O 134.11 NaH₂PO₄ * H₂O 62.5 ZnSO₄ * 7H₂O 0.43 glucose 4500HEPES 3575 hypoxanthine monosodium 2.4 linoleic acid 0.04 lipoic acid0.1 putrescine * 2HCl 0.08 sodium pyruvate 55 thymidine 0.4 d-biotin0.0037 calcium pantothenate 1.0 choline chloride 9.0 folic acid 2.7inositol 12.6 nicotinamide 2.0 pyridoxal HCl 2.0 pyridoxine HCl 0.031riboflavin 0.22 thiamine HCl 2.2 vitamine B12 0.68 F-68 1100 L-alanine4.6 L-arginine HCl 274 L-asparagine * H₂O 22.5 L-aspartic acid 20L-cysteine HCl * H₂O 17.56 L-cystine * 2HCl 52.29 L-glutamic acid 22L-glutamine 657 glycine 26.2 L-histidine HCl * H₂O 73.4 L-isoleucine 107L-leucine 111.4 L-lysine HCl 163.8 L-methionine 32.4 L-phenylalanine68.4 L-proline 17.25 L-serine 36.8 L-threonine 101 L-tryptophan 19.2L-tyrosine 91.7 L-valine 99.6 Osmolality 300 pH 7.1

Example 20 Assaying Viral Titers and High-Throughput Assay Techniques

The temperature-sensitive and wild-type adenovirus stocks used in thepreceding examples were produced in 293-1 cells in tissue cultureflasks. In this example, the levels of adenovirus being produced by293-1 cells was quantified by TCID₅₀ endpoint assay or infectivityassay.

The TCID₅₀ assay was conducted as follows: 1.0×10³ 293-1 cells wereplated into 96-well microtiter plates and infected with serial dilutionsof adenovirus stock and allowed to incubate at 37° C. in a humidified 5%CO₂ incubator. Eight replicates of 100 μl of each dilution wereinoculated onto the cells. Three days after infection the cells weremethanol fixed, washed with PBS and stained with FITC-conjugatedanti-hexon antibody (Biodesign) followed by propidium iodide staining tovisualize cell nuclei. After rinsing with PBS, the plate was examinedunder a fluorescent microscope and scored for the presence of hexoncontaining cells. Titer at endpoint was calculated using a Poissondistribution. A dilution of virus that yields 50% of replicate sampleshexon positive has 0.5 IU/100 μl inoculum. Infectious titer is theproduct of the reciprocal of this dilution, 0.5 IU/100 μl and 10(conversion factor to ml) to give the final infectious titer per ml.

A high-throughput microtiter infectivity assay to measure infectiousvirus was conducted as follows. Aliquots (10 μl) of serially dilutedcell-free supernatants were inoculated onto HeLa cells grown in 96-wellmicrotiter plates. After three days, infected cells were treated andlysed with a denaturation solution (addition of 1/10^(th) volume of 4.0M NaOH, 10 μg/ml salmon sperm DNA and 100 mM EDTA). Lysate wastransferred to a Silent Monitor BiodyneB plate (Pall) and vacuumfiltered onto the nylon membrane. The membrane was washed, denatured,hybridized with ³²P-labeled adenovirus E1A cDNA restriction fragment andanalyzed on a phosphorimager (Molecular Dynamics). Linear regressionanalysis of serially diluted adenovirus standards was used to calculateinfectious adenovirus titers in samples, using adenovirus standardstitered by the TCID₅₀ assay.

Specific virus productivity was calculated by normalizing infectiousvirus titers in the lysate to cell numbers at the time of infection.Results are shown in Table 14: TABLE 14 Adenovirus Production Specificproductivity Adenovirus Cell line (IU/cell) Assay Ad5 293-1 125 TCID₅₀HeLa S3 400 TCID₅₀ Ad5ts149 293-1 10 TCID₅₀ 293-1 16 microtiterinfectivity 293-1 15 microtiter infectivity 293-1 10 microtiterinfectivity

These results show that specific production of Adts149 in 293-1 cellswas one to two logs lower than Ads.

An Ad5 virus preparation of known titer showed a linear range extendingfrom 12.5 to 500 IU/well based on linear regression in the microtiterinfectivity assay.

Combining a viral infectivity assay with a microtiter array format asdescribed above resulted in a technique which is both rapid andquantitative, and which is highly suitable to automation.

The high-throughput infectivity assay as described above can also beapplied to assaying other viruses (e.g., rAAV and wtAAV). The assay canbe performed essentially as described above using appropriate mammaliancells (e.g., HeLa C37 cells for rAAV or 293 cells for wtAAV) and underconditions permissive for the replication of the virus to be assayed(e.g., in the presence of helper virus for rAAV and wtAAV); and thenlysates can be prepared and nucleic acids in said lysates can betransferred to a membrane as described above. Hybridization of themembrane containing the array of bound nucleic acid pools (each poolbeing released from the cells of the corresponding culture well) istypically performed with a suitable virus-specific probe (e.g., a probespecific for AAV rep and/or cap might be used to detect wtAAV, or aprobe specific for an inserted transgene might be used in the case of arecombinant AAV vector).

The above-described high-throughput infectivity assay exhibited a linearresponse in the determination of rAAV titers over a relatively broadrange of- concentrations. For example, when a viral preparation of knowntiter (as determined by a modified infectious center assay) was seriallydiluted 1:2, starting from 2400 infectious units or “IU”/well, and usedas a standard for the titer determination of two purified tgAAVCFpreparations of unknown titer each of which was serially diluted 1:5,the microtiter assay showed a linear range extending from 75 to 600IU/well based on linear regression. The determination of the titer ofwtAAV preferably employed a limiting dilution format (for example, wheneight serial limiting dilutions of a wtAAV preparation of known titerwere assayed, the titer determined by the microtiter assay wasessentially the same as that determined by the standard TCID₅₀ assay,3×10⁹ IU/ml).

Either with limiting dilution or by comparison to a known standard, aninfectious virus titer can be determined which corresponds to the titersdetermined by more classical techniques (e.g., the infectious centerassay or the TCID₅₀ 50% end-point analysis). Besides its use in thedetermination of viral titers, this high-throughput infectivity assayhas many others uses, including, but not limited to, the screening ofcell lines permissive or non-permissive for viral replication andinfectivity (e.g. by including various mammalian cells or variantsthereof in different wells of a microtiter array); as well as thescreening of agents that affect viral infectivity and/or replication(e.g. by including various agents in different wells of a microtiterarray as described above and determining the effect of the agents on theresulting infectious titer of virus). Among other things, the ability torapidly screen for agents or conditions that enhance viral infectivityand/or replication is particularly useful in the context of developingor optimizing the production of viral vectors. Conversely, the abilityto rapidly screen for agents or conditions that repress viralinfectivity/replication is quite useful in the context of identifyinganti-viral therapeutics.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

1-7. (canceled)
 8. A method of isolating a population of rAAV particles,comprising the steps of: (a) chromatographing an AAV producer celllysate containing rAAV particles on a positively-charged anion exchangeresin; and (b) chromatographing an AAV producer cell lysate containingrAAV particles on a negatively-charged cation exchange resin, whereby apurified population of rAAV particles is generated. 9-177. (canceled)